Biological measurement system

ABSTRACT

In a biological measurement system ( 10 ) for collecting one or more samples of sputum and/or mucus from a patient in the form of an aerosol and for analysing said one or more samples to detect whether or not pathogens are present therein, the one or more samples are in solution within the system ( 10 ) and detection of the pathogens is performed using a fluorescently labeled assay. The system ( 10 ) is adapted to detect bacterial pathogens using evanescent-wave spectroscopy preferably by using a single-reflective technique. The one or more samples are advantageously provided to the system ( 10 ) in aerosol form. However, the system ( 10 ) is capable of being adapted for use in analysing samples in liquid form. Methods of analysing said one or more samples in the system ( 10 ) are described.

FIELD OF THE INVENTION

[0001] The present invention relates to biological measurement systems;more particularly, but not exclusively, the invention relates to abiological measurement system for providing early detection ofrespiratory disease, for example tuberculosis induced by the pathogenmycobacterium tuberculosis.

REVIEW OF THE ART

[0002] Numerous biological measurement systems are known in the art fordetecting various forms of pathogens, for example bacteria, viruses,moulds and fungi.

[0003] In an article entitled ‘Detection of Antibody-Antigen Reactionsat a glass-liquid interface as a Novel Optical Immunoassay Concept’,Proceedings of 2^(nd) Optical Fibre Conference (Stuttgart 1984) pp. 75,R. M. Sutherland et al. describe an optic waveguide apparatus wherein anantibody species is covalently immobilized onto a surface of a planar orfibre-optic waveguide. A sample solution comprising an antigen ispresented to the surface, whereat the antigen is immobilized by theantibody species. The antigen is interrogated using an evanescent wavecomponent of a light beam, totally internally reflected many timeswithin the wave-guide. The evanescent component exhibits acharacteristic that it penetrates only a fraction of its wavelength intoan aqueous phase at the surface whereat the antigen is immobilized;thus, the evanescent component is capable of optically interacting withsubstances, for example the immobilized antigen, bound to or very closeto the interface and only minimally with any bulk solution which mayinterface onto the surface.

[0004] Moreover, in a published paper entitled ‘Sensitivity enhancementof evanescent wave immunoassay’ (1993) Yoshida et al. Meas. Sci.Technol. 4 pp. 1077-9, there is described a fluoro-immunosensor suitablefor the detection of low concentrations of pathogens in blood and serumsamples. The immunosensor employs an assay system including a sandwichassay in a flow cell. For a standard sandwich assay, a number ofwash-steps are required. These wash-steps complicate the system and makeit necessary for a relatively skilled operator to carry out the testing.

[0005] In a published United Kingdom patent no. GB 2174802, there isdescribed an optic-waveguide biosensor for detecting and monitoringspecific assay molecular species in flowing test fluid samples. Thebiosensor employs a complex multiple reflection optical geometry whereinfluorescence associated with the binding of an antigen to anantibody-coated surface is characterized by an increase in the signaldetected in the same direction as that detected for the multiplyreflected incident light. A disadvantage of this sensor is that bulkscattering by an optical waveguide forming a part of the sensor canaffect the signal level detected at the waveguide. Moreover, multilayerconstruction configurations of the optical waveguide serve to furthercomplicate the biosensor and the complexity of signal levels observed.Again, the operator of the biosensor must be relatively skilled.

[0006] In another published patent application no. GB 2227089, there isdescribed a system for the analysis of specific assay molecular speciesin test fluid samples. The system employs a detection method involvingthe use of detection of an evanescent wave component of an antibodyimmobilized on the surface of a planar or fibre optic waveguide. Thecoupling of the resonant wavelength is facilitated by an optical gratinglocated at either the interface between the dielectric body and themedium or between the dielectric body and the sensitized coating whichcan potentially result in alignment problems. Furthermore, the light isreflected many times within the waveguide and as such the intensityobserved is subject to losses due to scattering.

[0007] A European patent application no. EP 0519623 discloses anevanescent wave system comprising first and second wave propagatingsurfaces. The first surface is used to detect the presence of a firstanalyte and the second is used to indicate the presence of a secondanalyte and/or a reference. The system is complex and, in oneembodiment, makes use of the inner and outer surfaces of the two wavepropagating surfaces.

[0008] In a patent application no. EP 0239382, there is describedanother fibre optic based device that has a high numerical aperture anddoes not utilize any cladding at its contact points. The device is of acomplex design which employs a beam splitter and lens system susceptibleto scattering losses. Again, the device incorporates a flow cell for theinterrogation of optically-detectable assays. This device is ofrelatively high cost and complexity.

[0009] An international PCT application no. PCT/US01/21634 concerns anapparatus and method for evanescent light fluoroassays, specificallyintended for use on bodily fluids. The apparatus is designed to detectmultiple spatially resolved assays to be read simultaneously usingdetection sensors such as a CCD camera, a photodetector, a photoarray orrelated sensors. Air pressure, vacuum or capillary action is used tomove the sample onto an assay area of a disposable cartridge. Again,this apparatus utilizes multiple reflections and, in one embodiment,these reflections occur in a very thin film, which improves measurementsensitivity. The apparatus relies upon personnel performing tests totransfer samples onto a disposable element of the apparatus and as suchis not a ‘safe’ method of handling pathogenic samples. Moreover, thepersonnel are required to be of a high skill level and the apparatustests for multiple conditions which is not the principle performancerequirement of the biological measurement system of the presentinvention.

[0010] A U.S. Pat. No. 5,922,550 relates to a sensitive device for thedetection of immunoassays. The basis of the device is somewhat differentto that of the present invention in that the sensitive device uses apredetermined pattern of analyte specific receptors that, in thepresence of the pathogen, produce a diffraction pattern from transmittedor reflected light. A diffraction image thereby generated can beobserved by eye or by an optical reading device.

[0011] A microassay rod and card system is described in a U.S. Pat. No.4,673,657. The rod and card system is intended for the simultaneousdetection of the presence of numerous different biologically importantsubstances in a single small sample. The device is rapid and makes useof standard immunoassay systems that are well documented in theliterature. The sample must be placed on the card system and so a safemeans of collection is not provided. Moreover, the detection of multiplepathogens is not desirable in the conditions for which the measurementsystem of the present invention is intended.

[0012] In a U.S. Pat. No. 3,992,516, there is described a directfluorescent antibody composition and method for the detection ofpneumocystis carinii; the method of sample collection is not presentedin the patent.

[0013] A German patent application no. DE 3932784 is concerned with atest for analysing aerosols, including fluid from respiratory passagesand spittle. Exhaled breath is collected directly into a massspectrometer or is concentrated prior to analysis by collection onto acooled plate. Analysis by mass spectrometry leads to the determinationof the molecular species present in the gas/aerosol/liquid byfragmentation of these species and the subsequent determination of theirmasses.

[0014] Such an analysis results in a complex problem of co-addition todetermine all of the molecular species present. Collection on a cooledplate is a standard technique in the literature, namely matrixisolation, and has been utilized since the 1960's. The test relies ondirect measurement of the whole sample, rather than making use of achemical/biochemical assessment method The use of a mass spectrometer isexpensive and requires the incorporation of vacuum equipment; thus thistest is of high cost and is not portable.

[0015] In a United Kingdom patent application no. GB 2311856, anenvironmental sampler is described for recovering particles havingdiameters in a range of 0.1 μm to 20 μm. In the sampler, a feed is usedto coat the surfaces of beads in a bead bed with liquid, which thenentraps particles from an air sample. The liquid is then recovered andanalysed for the components that have dissolved in the liquid. Such anassay would not be appropriate for the collection of pathogens containedwithin sputum/mucus from the upper lung area.

[0016] In an international PCT application no. PCT/AU95/00540, there isdescribed a nasal/oral filter designed for particle entrapment byinhalation or exhalation. The filter comprises a collection systemdesigned to fit into a user's mouth or nostril and has a non-linear pathto capture particulates. In this filter, the main target particles forcapture are potentially allergenic species, but the possibility existsto capture viruses or mycobacteria. The particles can be recovered bywashing or blowing through the sample collection system and subsequentlyanalyzed by culture, nucleic acid analysis or similar processes. Such anapproach means that the sample is transferred to another system whichimmediately gives rise to safety issues concerning safe sample handlingand speed of testing the samples.

[0017] An international PCT patent application no. PCT/SE96/00474concerns a device that investigates one or more components of exhaledair for the presence of the pathogenic helicobacter pylori in thestomach and intestinal tracts of human beings. The device comprises atubular element for conducting exhaled air onto an airtight plateincorporating a porous membrane for sample collection. Prior toproducing the sample, the patient swallows an isotope-labeled,preferably radioactive, urea preparation, which breaks down in thepresence of helicobacter pylon. The preferred embodiment of the deviceindicates the presence of radioactive carbon dioxide formed as aconversion product of helicobacter pylori. The plate absorbs theradioactive carbon dioxide and is subsequently removed from the devicefor radioactive analysis. In the detection of viruses and bacteria, suchas mycobacterium tuberculosis, the device is inappropriate as there isno simple way provided to isotopically label the pathogen nor is thereany simple way of forming a simple breakdown product.

[0018] In a U.S. Pat. No. 4,350,507, there is described a particlesampling apparatus for the collection of dust particles from theatmosphere. The apparatus employs a grill and pre-filter system toremove largest non-respirable particles and then a main filter tocollect the respirable particles. This limits the dust concentration insome industrial situations to tolerable limits. The apparatus is notdisclosed as being capable of performing biochemical assay analysis.

[0019] In a U.S. Pat. No. 5,372,126, there is described a pulmonarysampling chamber designed for the safe collection of pulmonary samples.The apparatus entirely encloses the patient and, as such, is notportable. The primary aim of the system is to collect deep samplesecretions non-invasively from a patient's lungs. Moreover, the chamberis equipped with a replaceable exhaust filter unit to trap airbornepathogens and other harmful particles; the exhaust filter cansubsequently be removed for analysis and disposal.

[0020] A U.S. Pat. No. 3,745,991 describes a device for reducingenvironmental contamination during medical treatment and/or diagnosis.The aim of the device is to safely deliver and/or collect aerosolsamples from a patient by enclosing the patient's face and passing anyfluids generated by exhalation from the patient through a filter systemto collect any hazardous materials for later disposal/analysis. Thedevice is not easily portable.

[0021] The Problem(s) to be Solved Accordingly

[0022] It is becoming increasingly important for organisations, forexample government agencies and humanitarian relief agencies, to have attheir disposal facilities for the rapid detection and identification ofpathogens. Such pathogens include newly emerging viruses andre-emergence of known diseases such as bubonic plague, tuberculosis andcholera. On account of such pathogens becoming increasingly resilient tomedication, there is a considerable need for measurement systems thatcan be used for the early detection of pathogen outbreaks so thatisolation measures and targeted medication can be applied to containfurther pathogen spread.

[0023] Moreover, on account of outbreaks of disease often occurring ineconomically less advanced regions of the world and being spread byvectors such as aviation to other regions thereof, there is a need formeasurement systems which are relatively inexpensive, which arestraightforward to use by untrained staff, which can give results at thepoint-of-test/care, and which are potentially less susceptible toinadvertently spreading pathogens when operated by untrained staff.

[0024] In particular, the detection of bacterial infection is of vitalimportance in global terms. Field-testing of bacterial infection,preferably using a test that responds rapidly, is particularly desirablebecause of the prevalence, virulence and major impact of majorinfections such as pneumonia, tuberculosis, malaria and other pathogens.Contemporary bacterial tests are mainly based on complex laboratoryassays and are therefore potentially expensive and are not especiallysuitable for field use. Moreover, many contemporary tests require asubstantial time period, for example in a range of 2 to 4 weeks, toprovide a positive identification of the presence of pathogens. Morerecently, rapid tests have been developed which offer reducedidentification timescales to hours/days. These rapid tests are primarilybased on the analysis of sputum/mucus samples from upper lung regions;however, the collection and handling of such samples is hazardous topersonnel conducting and/or supervising such testing. Thus, bothtimescale and potential pathogen transmission problems make thesecontemporary tests difficult to execute in field locations.

[0025] Furthermore, at the current state of the art for thefield-testing of tuberculosis (TB), a ‘standard’ skin test employed iscompromised by HIV status and so the only method currently used in thethird world is that of smear microscopy on sputum/mucus samples. Theaccuracy of these tests is dependent on skilled operators and frequentre-tests.

[0026] In general, in these circumstances, a significantly safer methodof pulmonary bacterial sensing is required for addressing the pathogentransmission problems that are prevalent when collecting and handlingsamples for subsequent pathological analysis.

[0027] Furthermore, rapid and reliable detection of other types ofmedical condition, for example hormonal abnormalities in the context ofsteroid (hormone) abuse in professional sport, markers for cancer and soon is also highly desirable.

[0028] None of the known systems reviewed above adequately addressesthese problems.

SUMMARY OF THE INVENTION

[0029] According to a first aspect of the present invention, there isprovided biological measurement system for measuring the concentrationof components included in a sample, the system characterised in that itcomprises:

[0030] (a) collecting means for collecting the sample;

[0031] (b) concentrating means for spatially concentrating the sample;

[0032] (c) marking means for optically labeling the components presentin the concentrated sample; and

[0033] (d) interrogating means for optically interrogating the labeledcomponents and thereby generating a measure of the concentration ofcomponents present in the sample.

[0034] Preferably, the collecting means is adapted for collecting thesample in aerosol form. Aerosol sample collection is of benefit wherethe system is employed for testing pulmonary diseases and environmentalair-borne pollutants. It maximizes the pathogen collectable bycomparison with conventional (sputum-only or mucus-only) “cough”-stylemethods.

[0035] Preferably, the collecting means further comprises nebulizingmeans for emitting a mist for inducing aerosol emission from a subject.The nebulizing means is of benefit in that is capable of increasing theamount of sample obtained when performing pulmonary testing.

[0036] More preferably, the nebulizing means in use is adapted togenerate a saline mist comprising saline droplets having diameters in arange of 6 μm to 20 μm. The range is of advantage in that droplet sizesof less than 6 μm is too palatable to induce coughing whereas dropletsizes greater than 20 μm can be unpleasant to inhale.

[0037] Most preferably, the saline droplets have diameters in a range of10 μM to 15 μm and comprise saline solution having a salineconcentration in a range of 0.1% to 2% by weight.

[0038] Preferably, the concentrating means further comprises a featurefor scraping surfaces where the sample is deposited to spatiallyconcentrate the sample. Spatial concentration of the sample is capableof enhancing the measurement sensitivity of the system.

[0039] Preferably, the feature is elastically deformable for spreadingthe spatially concentrated sample over an optical interrogation regionwhereat the concentrated sample is subjected to optical interrogation.

[0040] Beneficially, the marking means comprises at least one of aselective binding assay and a competitive displacement assay foroptically marking presence of the components by way of fluorescentmarkers. Such assays are capable of providing effective interfacebetween biochemical and optical domains.

[0041] Preferably, the fluorescent markers are bound to antibodies foruse in at least one of the selective assay and the competitive assay.Antobodies, for example as employed in immunoassay, are of advantage inthat they can be made highly selective with remain to moleculargroupings or microbes to which they are capable of binding. Moreover,antibodies are presently becoming relative cheap to manufacture in bulkusing contemporary genetic engineering processes.

[0042] Preferably, the fluorescent markers are fluorophores bound to theantibodies by way of an intermediate carrier such that a plurality offluorophores are associated with each antibody. The use of fluorophoresis of advantage in that they are capable of being excited by opticalradiation at a first radiation frequency and emitting fluorescentradiation at a second radiation frequency, the first and secondfrequencies being mutually different, thereby enabling the excitationradiation and the emitted fluorescent radiation to be individualisolated.

[0043] More preferably, the intermediate carrier is implemented in theform of latex spheres.

[0044] The interrogating means beneficially comprises an opticalevanescent detector for detecting changes in optical response induced bythe presence of the components. Evanescent wave interrogation isespecially advantageous as it enables a planar optical surface acrosswhich the sample is spread to be specifically targeted forinterrogation.

[0045] Preferably, the evanescent detector includes:

[0046] (a) one or more of a diode laser and a LED as a source ofinterrogating radiation for interrogating the concentrated sample; and

[0047] (b) one or more of a avalanche photodiode, a photodiode array anda photomultiplier tube as an optical detector for detecting fluorescentradiation emitted from the concentrated sample in response to opticalinterrogation of the sample, the optical detector for generating adetection signal indicative of changes in fluorescence from the sampleresulting from the presence of the components in the sample.

[0048] Such sources and detectors of optical radiation are of advantagein that they are potentially inexpensive, compact and robust.

[0049] Preferably, the system further comprising strobing means forstrobing radiation emitted from the source of interrogating radiation,and synchronous demodulating means for demodulating the detection signalin synchronism with the strobe to render the system less sensitive toquasi-constant optical radiation received at the optical detector. Suchstrobing is capable of rendering the system less influenced by theeffects of stray ambient illumination penetrating into the system.Moreover, such a strobe also enables effects of offset voltages withinelectronic components of the detecting means on the measurement to besignificantly reduced.

[0050] Preferably, the system further comprising computing means forchanges in the detection signal when the components in the sample areoptically labeled or displace optical labels.

[0051] More preferably, the computing means is arranged to monitor theconcentrated sample before and after fluorescent labeling thereof tocalculate the measure of the concentration of the components in thesample. Such dual measurement is of benefit in removing effects ofsystematic errors in the system, for example background fluorescenceoccurring in the interrogating means.

[0052] Beneficially, the computing means further comprises one or moreof:

[0053] (a) displaying means for displaying the measure of theconcentration of the components in the sample, and

[0054] (b) data logging means for storing a record of measure of theconcentration of the components.

[0055] Preferably, the collecting means is arranged to enclose thesample, thereby preventing personnel contact with the sample when thesystem is in use. Such containment is of advantage in assisting toprevent the spread of dangerous pathogens and also renders the systemsafer in use.

[0056] More preferably, the collection means is arranged to be asingle-use disposable part. Such single-use is of further advantage inpreventing the spread of potentially dangerous pathogens. Mostpreferably, the collecting means comprises features rendering itsubstantially undismantleable after sample collection therein.

[0057] Preferably, the collecting means comprises vortex enhancing meansfor deposition of the sample within the collecting means.

[0058] The collecting means preferably comprising filtering means for atleast partially inhibiting spread of the components of the sample fromthe collecting means.

[0059] Preferably, the marking means includes lysing means for causinglysis of the components present in the sample, thereby enhancingmeasurement sensitivity of the system by increasing the number ofavailable potential optical labeling sites.

[0060] The system according to the first aspect is capable of being usedin a wide range of applications not limited to the biological domain. Inparticular, but not exclusively, the system is preferably adapted toidentify the components in the form of one or more of the following:

[0061] (a) antibodies;

[0062] (b) nucleic acids;

[0063] (c) enzymes and/or other proteins;

[0064] (d) analogues of one or more of (a) to (c); and

[0065] (e) a microorganism.

[0066] With regard to microorganisms, the system is especiallyappropriate for the detection of one or more of the following:

[0067] (a) a virus;

[0068] (b) spores;

[0069] (c) molds;

[0070] (d) pollen; and

[0071] (e) a microbiological allergen.

[0072] Moreover, the system is also preferably adapted to identify thecomponents in the form of one or more of the following:

[0073] (a) toxic dust;

[0074] (b) an explosive;

[0075] (c) a drug; and

[0076] (d) a pollutant.

[0077] According to a second aspect of the present invention, there isprovided a method of detecting one or more pathogens in one or moresamples of sputum from a subject using a system according to the firstaspect of the invention, the method involving the steps of:

[0078] (a) collecting said one or more samples in the collecting means;

[0079] (b) spatially concentrating the one or more samples in theconcentrating means;

[0080] (c) optically labeling one or more pathogens present in said oneor more samples;

[0081] (d) optically interrogating the pathogens to achieve an opticalresponse; and determining from the optical response of said one or moresamples whether or not said one or more pathogens are present in saidone or more samples.

[0082] Preferably, in steps (b) and (c), a fluorescently labeled assayis employed to provide the optical response.

[0083] Preferably, in steps (b), (c) and (d), detection of fluorescenceis performed using evanescent-wave spectroscopy.

[0084] Preferably, when executing the method, said one or more pathogenscomprise one or more of:

[0085] (1) antibodies;

[0086] (2) nucleic acids;

[0087] (3) enzymes or other proteins;

[0088] (4) analogies of (1) to (3); and

[0089] (5) a micro-organism.

[0090] The method is advantageously adapted for the detection ofbacteria associated with pulmonary and pulmonary-related infections.

[0091] Moreover, the method is preferably adapted for the detection ofone or more of the following pathogens:

[0092] (1) a virus;

[0093] (2) a protein and/or antibody;

[0094] (3) another symptomatic particle not included in (1) or (2);

[0095] (4) a spore;

[0096] (5) a mold;

[0097] (6) pollen;

[0098] (7) an allergen;

[0099] (8) toxic dust;

[0100] (9) an explosive;

[0101] (10) a drug; and

[0102] (11) a pollutant.

[0103] Preferably, to enhance aerosol generation, the inhalation of oneor more of:

[0104] esters, water vapour, saline vapour, expectorant and menthol isused to assist release of bacteria-containing mucus from the trachea orfrom the upper lung of a subject being tested.

[0105] Beneficially, a partial negative pressure is employed to assistin obtaining said one or more samples in aerosol form.

[0106] The method is capable of being applied to testing a diverse rangeof samples. For example, said one or more samples preferably comprise anaerosol of blood or other bodily fluid or bodily fluid in liquid form.

[0107] In the method, analysis of said one or more samples is performedusing one or more of:

[0108] (a) an ELISA chromogenic reaction; and

[0109] (b) a surface acoustic wave (SAW) biosensor to detect an antigenin said one or more samples.

[0110] According to a third aspect of the present invention, there isprovided a sample collection apparatus or collecting aerosol samples,characterised in that the apparatus comprises:

[0111] (a) collecting means for collecting the sample; and

[0112] (b) concentrating means for spatially concentrating the sample.

[0113] Preferably, the collecting means further comprises nebulizingmeans for emitting a mist for inducing aerosol emission from a subject.

[0114] Preferably, the nebulizing means in use is adapted to generate asaline mist comprising saline droplets having diameters in a range of 6μm to 20 μm.

[0115] More preferably, the saline droplets have diameters in a range of10 μm to 15 μm and comprise saline solution having a salineconcentration in a range of 0.1% to 2% by weight.

[0116] Beneficially, the concentrating means further comprises a featurefor scraping surfaces where the sample is deposited to spatiallyconcentrate the sample.

[0117] Preferably, the feature is elastically deformable for spreadingthe spatially concentrated sample over an optical interrogation regionwhereat the concentrated sample is subjected to optical interrogation.

[0118] Preferably, the apparatus further comprises an interrogationregion whereat the sample in spatially concentrated, the interrogationregion being susceptible to optical interrogation.

[0119] Preferably, the interrogation region is susceptible to evanescentwave interrogation.

[0120] Preferably, the collecting means is arranged to enclose thesample, thereby preventing personnel contact with the sample when theapparatus is in use.

[0121] To reduce the potential spread of dangerous pathogens, thecollecting means preferable comprises features rendering itsubstantially undismantleable after sample collection therein.

[0122] Preferably, the collecting means comprises vortex enhancing meansfor deposition of the sample within the collecting means. Vortexenhancing means include one or more of a septum and a bend in a samplecollection region.

[0123] To reduce the risk of spreading potentially dangerous pathogens,the collecting means preferably comprises filtering means for at leastpartially inhibiting spread of the components of the sample from thecollecting means

[0124] According to a fourth aspect of the present invention, there isprovided an immunosensor for collecting one or more samples of sputumfrom a patient in the form of an aerosol and for analysing said one ormore samples to detect whether or not pathogens are present therein.

[0125] The immunosensor is capable of providing a significantly safermethod of pulmonary testing, the sensor being designed for safe handlingof test samples.

[0126] Preferably, in the immunosensor, said one or more samples are insolution within the sensor and detection of the pathogens within saidone or more samples is performed using a fluorescently labeled assay.The fluorescently labeled assay is capable of providing a sensitive andreliable approach to detecting presence of the pathogens.

[0127] More preferably, the detection of bacterial pathogens isperformed using evanescent-wave spectroscopy or fluorimetry. The use ofevanescent-wave spectroscopy or fluorimetry enables opticalinterrogation to be applied efficiently to a relative small sample ofpathogen to detect its presence.

[0128] Preferably, the immunosensor is adapted to detect one or more ofthe following pathogens:

[0129] (a) antibodies;

[0130] (b) nucleic acids;

[0131] (c) enzymes and/or other proteins;

[0132] (d) analogues of one or more (a) to (c); and/or

[0133] (e) a micro-organism.

[0134] More preferably, the immunosensor is adapted for the detection ofbacteria in a sample, the bacteria being associated with pulmonary andpulmonary-related infections. Alternatively, or additionally, theimmunosensor is adapted for the detection of one or more of:

[0135] (a) a virus;

[0136] (b) a protein and/or antibody;

[0137] (c) other symptomatic particles not included in (a) and (b); forexample indicators of forms of cancer.

[0138] (d) spores;

[0139] (e) molds;

[0140] (f) pollen;

[0141] (g) an allergen;

[0142] (h) toxic dust;

[0143] (i) an explosive;

[0144] (j) a drug; and

[0145] (k) a pollutant.

[0146] According to a fifth aspect of the present invention, there isprovided a method of detecting one or more pathogens in one or moresamples of sputum from a patient using an immunosensor according to thefourth aspect of the invention, the method involving the steps of:

[0147] (a) collecting said one or more samples in the immunosensor;

[0148] (b) fluorescently labeling one or more pathogens present in saidone or more samples;

[0149] (c) interrogating said one or more samples using opticalinterrogation to achieve an optical response; and

[0150] (d) determining from the optical response of said one or moresamples whether or not said one or more pathogens are present in saidone or more samples.

[0151] Preferably, in steps (b) and (c), a fluorescently labeled assayis employed to provide the optical response.

[0152] More preferably, for efficiently interrogating a relatively smallquantity of sample, detection of fluorescence in steps (b), (c) and (d)is performed using evanescent-wave spectroscopy or evanescent-wavefluorimetry.

[0153] Preferably, the method is susceptible to detecting the occurrenceof said one or more pathogens by way of:

[0154] (1) antibodies;

[0155] (2) nucleic acids;

[0156] (3) enzymes or other proteins;

[0157] (4) analogies of (1) to (3); and

[0158] (5) a microorganism.

[0159] More preferably, the method is adapted for the detection ofbacteria associated with pulmonary and pulmonary-related infections.

[0160] Alternatively, or additionally, the method is preferably adaptedfor the detection of one or more of the following pathogens:

[0161] (1) a virus;

[0162] (2) a protein and/or antibody;

[0163] (3) another symptomatic particle not included in (1) or (2);

[0164] (4) a spore;

[0165] (5) a mold;

[0166] (6) pollen;

[0167] (7) an allergen;

[0168] (8) toxic dust;

[0169] (9) an explosive;

[0170] (10) a drug; and

[0171] (11) a pollutant.

[0172] In order to induce more efficient sample generation, the methodpreferably involves the inhalation of one or more of:

[0173] esters, water vapour, saline vapour, expectorant and menthol toassist release of bacteria-containing mucus from the trachea or from theupper lung of the patient. The patient, in this case, may be either ahuman being or an animal.

[0174] Inhalation of the vapour should be from a separate vessel suchas, but not exclusively, a simple nebuliser, from within the samplecollection system or via an inlet tube to the sample collection system.Inhalation may take place via a pipe which may or may not incorporate ademand valve, diaphragm valve or similar.

[0175] Exhalation may be via a ‘plug’ in the pipe that ruptures to allow‘breath’ to enter the chamber and activate the fluorophore markedantibody.

[0176] The sample is collected directly onto a prism in the samplecollection system.

[0177] Exhalation by the patient is via a large inlet pipe. The aerosolexits the sample collection system via a smaller diameter pipe, into afilter or sample collection bag. The effect of the two differentdiameters is the creation of a ‘swirl’ effect in the vessel.

[0178] Exhalation into the sample collection vessel may be via a plugthat ruptures to release a hydrating agent, such as PBS or water, and/orthe desired antibodies and/or fluorescent markers into the samplecollection vessel. Further, exhalation into the sample collection vesselmay be via a pipe or pipe containing a venturi. The exit pipe of thesample collector may also incorporate a venturi.

[0179] A partial negative pressure may be employed to assist inobtaining said one or more samples in aerosol form.

[0180] Preferably, said one or more samples comprise an aerosol of bloodor other bodily fluid, such as urine, pathogenic sera, semen, saliva,tears or sweat. The analysis of these fluids significantly increases therange of pathogens that can be tested. Tests on saliva can, for example,be carried out to detect streptococcus and staphylococcus.

[0181] The method is preferably adapted to analyse one or more samplesof non-biological origin.

[0182] The interrogation technique is also adapted for use to detect allof the pathogens described above, with liquid samples of bodily fluidssuch as blood, urine, pathogenic sera, semen, saliva, tears or sweat andother samples such as food and non-biological samples.

[0183] The samples, either in the form of aerosol or liquid, may bediluted using PBS, water or other appropriate solvent.

[0184] The method is preferably adapted to cope with particles ofnon-biological origin including at least one of an environmental aerosoland an effluent.

[0185] More preferably, analysis of said one or more samples isperformed using one or more of:

[0186] (a) an ELISA chromogenic reaction; and

[0187] (b) a surface acoustic wave (SAW) biosensor to detect an antigenin said one or more samples.

[0188] Preferably, the sensor is used to execute a test which analysesexhaled breath, which is in the form of an aerosol, comprising bacteriaor other pathogens to be detected contained in water or sputum dropletsof such breath. The aforesaid one or more samples are preferablycollected directly into a sample tube for testing using, for example, afluorometric assay.

[0189] Hydration, using PBS, water or other suitable solvent may berequired in order to differentiate between bulk and surfacefluorophores.

[0190] A fluorometric assay technique may require time for culture toincrease the sample numbers to aid detection.

[0191] Preferably the sample collection system will be shatterable orsplinterable after use for safe disposal after a single use.

[0192] Fluorimetry has been shown to be of considerable importance forthe detection of biological materials such as proteins and DNA, wherefluorophores on antibodies are used as markers for detection. Detectionusing such fluorimetry can be executed by way of either

[0193] (a) bulk fluorescence measurements; or

[0194] (b) through the application of interrogation techniques such asevanescent wave detection; or

[0195] (c) cavity ring down spectroscopy; or

[0196] (d) through the use of displacement assays.

[0197] Such fluorimetry offers some potential advantages in terms ofspecificity, simplicity, and sensitive. Evanescent wave detection iswell known, but low-cost evanescent wave fluorimeters are not yetcommercially available for use in pathogen detection as described withrespect to the present invention.

[0198] The inventors are unaware of prior art regarding developments onthe detection of pulmonary bacterial infectious agents from exhaledbreath using fluorimetry. The present invention represents animprovement over exiting techniques because it reduces the level ofexpertise required to carry out an assay test; moreover, hazardassociated with potential transmission of diseases to the tester fromhandling contaminated samples is reduced. The inventors have thereforedevised an immunosensor which is fast acting, low cost, and portablewith disposable sample holders; the immunosensor is especiallysusceptible to use in field environments, for example in third-worldcountries. It is designed for screening large patient numbers andretesting as appropriate in for example, schools and institutions.

[0199] In the sixth aspect present invention, there is provided a samplecollection apparatus comprising:

[0200] (a) a sample collection volume bounded by an interior surface forreceiving a gaseously-borne sample;

[0201] (b) A sample collection volume bounded by an interior surface forreceiving a liquid sample where the liquid is sprayed into or addeddropwise into the sample collector, and

[0202] (c) collecting means for collecting, in use, at least a portionof the sample deposited on the interior surface and for concentratingthe portion at a test location susceptible to subsequent interrogation.

[0203] The invention is of advantage in that the apparatus is capable ofeffectively and conveniently collecting the sample for analysis.

[0204] Preferably, the collection volume is provided with vortexgenerating means for causing, in use, an incoming jet transporting thegaseously-borne sample to form into one or more vortices to assist withdeposition of the sample onto said interior surface.

[0205] Vortex flow in a fluid carrying a particulate load results inconversion of kinetic energy in the flow to thermal dissipation thereinand a subsequent deceleration with a resulting deposition of theparticulate load transported within the flow.

[0206] Preferably, the collection volume is implemented as a tubularelement and the collecting means is implemented as a plunger elementarranged to slidably engage within the interior surface of the tubularelement. Such an arrangement is of advantage in that the tubular elementis convenient for offering to users' mouths and for hand-held support,whereas the plunger element is capable of sealing an end of the tubularelement and, when pushed into the tubular element, assisting tospatially concentrate the sample.

[0207] More preferably, the plunger element forms a sufficient seal ontothe tubular element for collecting the sample into a ring-like mass whenthe plunger element, in use, is slidably moved within the tubularelement.

[0208] Preferably, the plunger element includes an end region comprisinga projection susceptible to collecting the ring-like mass together whenthe plunger element is rotated relative to the tubular element. Theprojection is capable of functioning in a spoon-like manner to scoop upthe sample from the tubular element to concentrate it into one spatiallocation.

[0209] The plunger element advantageously includes at its end region,optical interfacing means for interfacing between optical interrogatingmeans and the sample, thereby enabling the optical interrogating meansto interrogate the sample via the optical interfacing means. Use ofoptical interrogating means is of benefit in that non-contactinterrogation of the sample can be achieved, thereby, in the case ofcontagious pathogens, reducing the risk of spreading disease further.

[0210] Preferably, the plunger element comprises a hollow interiorregion for receiving, in use, the optical interrogating means.Concentric mounting of the interrogating means within the plungerelement, and concentric mounting of the plunger element within thetubular element is of benefit in enabling the interrogating means to bebrought in close proximity, for example within a few mm, of the sample.Moreover, such concentric mounting also renders the apparatuspotentially highly compact.

[0211] Preferably, the tubular element and the plunger element aredesigned to be disposable items whereas the optical interrogating meansis designed to be a non-disposable item. Such disposability is ofadvantage when the tubular element and the plunger element are used tocollect samples including pathogens that are potentially contagious; thetubular element and the plunger element can, for example, be disposed ofby incineration to circumvent spread of undesirable pathogens. Morepreferably, the tubular element and the plunger element are designed tobe mutually interlocking after sample collection has occurred therein toprevent these elements being reused with associated risk ofcross-contamination.

[0212] The optical interfacing means preferably comprises a prism forguiding interrogating radiation from the interrogating means to thesample, and for guiding response radiation from the sample back to theinterrogating means. Use of a single optical component forbi-directional optical radiation propagation enables the cost and sizeof the apparatus to be potentially reduced. More preferably, the prismis a dove-type prism; such a prism is susceptible to being used, forexample, in evanescent-wave optical interrogation of samples, especiallysamples subjected to fluorophore tagging.

[0213] The interrogating means comprises a source of strobed radiationfor providing the interrogating radiation, and a photodetector andassociated demodulator for detecting response radiation from the sampleand for demodulating the response radiation with respect to the strobe.Such a strobe arrangement can be applied to discriminate ambientquasi-constant optical radiation contributions, for example as a resultof light leakage into the apparatus from its ambient environment.

[0214] Preferably, for ease and cheapness of manufacture, one or more ofthe tubular element and the plunger element are fabricated from plasticsmaterials. More preferably, the plastics materials comprise one or moreof an acrylate, polyethylene, polypropylene, silicone rubber, polyvinylchloride (PVC), alkylene, polycarbonate, and polytetrafluoroethylene(PTFE) plastics material. Most preferably, the plastics materials areinjection moulded.

[0215] According to a seventh aspect of the invention, there is provideda method of collecting a sample from a user utilizing an apparatusaccording to the first aspect of the sample collection system, themethod comprising the steps of:

[0216] (a) exhaling mucus droplet borne air from the user into acollection volume of the apparatus;

[0217] (b) depositing mucus droplets from the exhaled air onto aninterior surface of the collection volume;

[0218] (c) collecting the droplets together from the surface usingcollecting means of the apparatus to provide a collected mass ofdroplets.

[0219] Preferably, the method further comprising the step ofinterrogating the collected mass of droplets after step (c) to determineone or more characteristics thereof. More preferably the collected massis interrogated optically.

[0220] Preferably, in the method, the collected mass is arranged tofluoresce in response to being optically interrogated, and the one ormore characteristics determined from the fluorescence.

[0221] Preferably, in step (b) of the method, the exhaled air isarranged to flow in vortices to promote deposition of the droplets ontothe interior surface.

[0222] It will be appreciated that features of the invention describedin the foregoing can be combined in any workable combination fallingwithin the scope of the invention as defined by the final claims.

DESCRIPTION OF THE DRAWINGS

[0223] Embodiments of the invention will now be described, by way ofexample only, with reference to the following diagrams in which:

[0224]FIG. 1 is a schematic diagram of a biological measurement systemaccording to the invention;

[0225]FIG. 2 is a schematic illustration of operation of a samplecollection unit of the measurement system of FIG. 1:

[0226]FIG. 3 is an illustration of a sample collection tube of themeasurement system of FIG. 1;

[0227]FIGS. 4a to 4 c are illustrations of a plunger suitable for usewith the collection tube of FIG. 3;

[0228]FIG. 5 is an illustration of an electronics module of a readerunit of the measurement system of FIG. 1;

[0229]FIG. 6 is an illustration of alternative sample collection tubesfor the measurement system of FIG. 1;

[0230]FIG. 7 is a schematic diagram of a yet further alternative samplecollection tube for the measurement system of FIG. 1;

[0231]FIGS. 8a and 8 b are illustrations of optical components includedwithin the electronics module of FIG. 5;

[0232]FIG. 9 is a schematic diagram of a sealing cap included within thesample collection unit of FIG. 2;

[0233]FIG. 10 is a schematic representation of an optical configurationemployed within the measurement system of FIG. 1;

[0234]FIG. 11 is an illustration of a modification to the measurementsystem of FIG. 1 for the analysis of liquid samples such as blood;

[0235]FIG. 12 is a schematic diagram of a compact dove prism forincorporation into the measurement system of FIG. 1;

[0236]FIGS. 13 and 14 are depictions of a selective binding assayemployed in the system of FIG. 1; and

[0237]FIG. 15 is a depiction of a competitive displacement assayemployed in the system of FIG. 1.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0238] In the following description, embodiments of a biologicalmeasurement system will initially be described in overview. Later,component parts of the embodiments and their associated biochemistrywill be described in more detail.

[0239] The system described herein employs evanescent wave spectroscopyand evanescent wave fluorimetry to detect the presence of a pathogenicsubstance using an immunoassay technique.

[0240] 1. System Overview

[0241] Referring firstly to FIG. 1, there is shown a biologicalmeasurement system according to the invention. The system is indicatedgenerally by 10 and comprises a sample collection unit indicated by 30,and a corresponding complementary reader unit indicated by 50. Fordisplaying test results, the reader unit 50 includes a readout display60. The collection unit 30 is adapted for collecting exhaled materialfrom a user 40, such material providing test samples for subsequentanalysis.

[0242] The collection unit 30 is designed to engage mechanically intothe reader unit 50. Moreover, the collection unit 30 is sufficientlycompact for it to be hand-held by the user 40. Furthermore, thecollection unit 30 is implemented in the form of a hollow sample tube 70comprising:

[0243] (a) an input orifice 80 for engaging onto a mouth region of theuser 40;

[0244] (b) an intermediate orifice 90 for coupling to a gas collectingregion, for example to an inflatable bag 100; and

[0245] (c) an access orifice for a piston-like plunger 110 which isslidably and rotationally moveable within the sample tube 70.

[0246] In order to reduce a risk of cross-contamination from one user toanother, the collection unit 30 is designed to be a disposable item;namely, the collection unit 30 is used only once to collect the samplefor testing and to safely present the sample to the reader unit 50 forinterrogation. The collection unit 30 is preferably molded from aplastics material to render it relatively inexpensive to manufacture,and also to render it susceptible to incineration to reduce the spreadof potentially dangerous pathogens collected therein. Moreover, thecollection unit 30 is designed so that the reader unit 50 is preventedfrom coming into direct contact with collected samples within the tube70 which can potentially comprise dangerous pathogens.

[0247] 2. Overview of System Operation

[0248] Operation of the biological measurement system 10 will now bedescribed in overview with reference to FIGS. 1 and 2.

[0249] After manufacture including the deposition of activebiomaterials, the collection unit 30 is preferably sealed within adesiccated hermetically-sealed package for storage prior to deployment.Such a package potentially prevents moisture from denaturing theaforementioned active biomaterials and also potentially reduces the riskof the collection unit 30 unintentionally becoming contaminated withpathogens prior to use; thus, such a package assists to prevent thesystem 10 from yielding unrepresentative test results.

[0250] Step 1: Immediately prior to deployment, the user 40, or a personsupervising testing, removes the collection unit 30 from its hermeticpackage. The user 40 then engages his/her mouth to the input orifice 80of the sample tube 70.

[0251] Step 2: Next, if required to assist in the production of a samplefrom the user 40, a saline mist is generated either from within thesample tube 70, for example from a miniature pressurized gas canisteratomizer coupled thereto, or within a nebulising device remotelyconnected to the collection unit 30; conveniently, the nebulising deviceis a foot-operated pump-like device. The user 40 inhales the saline mistvia the input orifice 80, the mist inducing sufficiently vigorouscoughing for the user 40 to exhale sputum and/or mucus in the form of anaerosol through the orifice 80 into the sample tube 70. The aerosolpasses into the tube 70 and is encouraged by the aerodynamic internalprofile of the tube 70 to circulate and decelerate in a vortex-liketrajectory to deposit mucus and/or sputum onto internal walls of thetube 70. Preferably, a collection volume, for example the inflatable bag100 of plastics material such as polyethylene or polyvinyl chloride(PVC), connected to the intermediate orifice 90 is included to receiveair exhaled by the user 40; each cough can amount to two litres volumeof air, hence the bag 100 is conveniently sized to accommodate severalcoughs. More preferably, the collection bag 100 is provided with an gasexit orifice 105 comprising a fine filter having a pore size which issufficiently large to enable the bag 100 to deflate over a period ofseveral tens of seconds, thereby rendering the bag 100 subsequentlyconvenient in size to handle when deflated, but also sufficiently smallto substantially prevent spread of potential pathogens exhaled by theuser 40. Moreover, the intermediate orifice 90 is of moderate flowresistance relative to the input orifice 80 and exit orifice 105 and ispreferably included between the sample tube 70 and its associated bag100 so as to enhance the aforesaid vortex gas trajectory and promoteefficient deposition of mucus and/or sputum within the sample tube 70.

[0252] Alternatively, a sample of saliva can be collected from the testsubject via the action of spitting into the sample collection apparatus.

[0253] Step 3: When a sufficiently large sample of sputum and/or mucusis collected within the collection unit 30, a sealing cap (not shown inFIG. 1, but denoted by 900 in FIG. 2) is placed over the input orifice80. Next, the plunger 110 is actuated to mechanically concentrate thesputum and/or mucus within an interrogation region, for example anoptical surface 120 of the plunger 110; in particular, the sputum and/ormucus is concentrated onto the optical interrogation surface 120provided at an end face of the plunger 110, the optical surface 120being capable of supporting evanescent interrogation-radiationpropagation which will be described in more detail later. Preferably,the plunger is both pushed and rotated within the sample tube 70 tomechanically concentrate the test sample at the surface 120. Morepreferably, the plunger 230 is rotated by at least 360° to ensure thatas much of the sample as possible is collected onto a sample collectionprojection of the plunger.

[0254] An incubation period may be needed prior to opticallyinterrogating the sample to generate a measurement reading.

[0255] Step 4: When the plunger 110 has been pushed substantially fullyinto the sample tube 70 to fully collect the sample onto the opticalsurface 120, the collection unit 30 is then offered to the reader unit50 so that a projection 130 thereof couples into the plunger 110 toenable optical interrogation of the optical surface 120 for determiningoptical properties of the sample thereat; such optical interrogation ispreferably achieved by way of evanescent light propagation at thesurface 120. Results of the optical interrogation are presented on thedisplay 60 to the user 40 and/or associated tester to establish whetheror not the user 40 is infected with one or more pathogens, for examplemycobacterium tuberculosis, to which the system 10 is responsive.

[0256] This completes an overview of operation of the system 10.

[0257] One or more of the sealing cap, the collection tube 70 and itsplunger 110 can have incorporated therein one or more reservoirs ofliquid for treating the optical surface 120 prior to opticalinterrogation thereof. Such puncturable reservoirs preferably containbuffer solutions or reagents such as, but not exclusively:

[0258] (a) lysing agents for causing collected pathogens, for examplemycobacterium, to fragment;

[0259] (b) rinsing agents for rinsing displaced fluorophores from theoptical surface 120 and/or flooding the optical surface 120 influorophores coupled to pathogen-selective antibodies;

[0260] (c) thinning agents to break up mucus; and

[0261] (d) developing agents for the sample such as labeled antibodies.

[0262] Reagents may be in the form of solids such as a lyophilizedsphere to protect them during storage; such solid reagents may bepresent in one or more reservoirs or in the sample collection tube 70.

[0263] These one or more reservoirs are preferably arranged to be userpuncturable to deliver their contents after sample collection but priorto optical interrogation. Mechanical construction of the reservoirs willbe described later with reference to FIG. 9.

[0264] As will be further described later, the optical surface 120 isone optical face of a prism configured to support evanescent lightradiation propagation therealong. The prism is preferably implemented asa dove prism, although alternative types of prism can be employed.

[0265] 3. System Component Parts

[0266] Detailed design of individual components of the system 10 willnow be described.

[0267] 3.1 Sample Collection Unit

[0268] Referring next to FIG. 3, there is shown the hollow sample tube70 implemented as a substantially cylindrical hollow sample tube 200.The tube 200 comprises a first open end indicated by 210 for receivingan exhaled sample from the user 40; the first open end 210 correspondsto the input orifice 80 in FIG. 1. Moreover, the tube 200 furthercomprises a second end 220 for receiving a hollow plunger 230; theplunger 230 corresponds to the plunger 110 of FIG. 1. The tube 200 alsoincludes a substantially cylindrical side tube 240 serving as theintermediate orifice 90, the side tube 240 having an associatedlongitudinal central axis substantially orthogonal to that of the sampletube 200. At a region where the tubes 200, 240 adjoin, there ispreferably included a mesh or filter gauze 250. The tube 200 furthercomprises a peripheral ring 260 around the first end 210 so that thisend 210 is substantially devoid of any sharp edges which could injurethe user 40 manipulating the tube 200, for example when the user 40manipulates the tube 200 towards his/her mouth.

[0269] The hollow plunger 230 is also of substantially cylindrical formand fabricated to be slidably and rotationally moveable concentricallywithin the inside of the sample tube 200 as illustrated, the tube 200and the plunger 230 being a mutually precise fit. Preferably, theplunger 230 is provided with a resiliently deformable sealing ring (notshown) substantially at an end of the plunger 230 offered to the sampletube 200 when in use. The sealing ring is preferably fabricated from anitrile rubber material, for example proprietary Viton material,silicone or polytetrafluoroethylene (PTFE). Moreover, the sealing ringis advantageously devoid of any lubricating material, for examplesilicone grease, which could potentially contaminate samples collectedwithin the tube 200, and thereby compromise system 10 operation.Additionally, vapour emitted from a lubricant may potentially be harmfulto the user 40 if ingested.

[0270] The hollow tube 200 is additionally provided with a salineatomizing assembly indicated generally by 300 at a region of the tube200 near to the first open end 210. The assembly 300 is preferablycoupled to a nebulizer, for example a foot-pump operated device, forforcing saline solution at pressure to the assembly 300 for generating adivergent jet 310 of saline mist for inhalation by the user 40; thesaline mist is effective at promoting vigorous coughing to induce user40 ejection of sputum and/or mucus. Preferably, the jet 310 comprisessaline droplets having a diameter in a range of 6 μm to 20 μm. Morepreferably, the saline droplets have a diameter in a range ofsubstantially 10 μm to 15 μm. A saline solution from which the dropletsare generated preferably is of a concentration in a range of 0.1% to 2%by weight of sodium chloride to water; more preferably, the salinesolution is of a concentration in a range of 0.7% to 1.1% by weight. Theassembly 300 includes a substantially central capillary tube 320 whichis angled at its nozzle end towards the first open end 210 to reduce anamount of saline mist swept towards the plunger 230. Preferably, theassembly 300 is recessed relative to the inside bore of the hollow tube200 so that the plunger 230 can be advanced towards the first end 210beyond a region where the assembly 300 is connected to the hollow tube200 as illustrated. The assembly 300 is preferably integrally molded aspart of the hollow tube 200; alternatively, in order to simplify moldingtools required, the assembly 300 can be a snap-fit retained insert whichis assembled into a projecting side port of the hollow tube 200 duringmanufacture. If required, the side port can be molded with its centralaxis orientated towards the first end 210 so that the insert does notrequire its capillary tube 320 to be shaped towards this end 210.

[0271] Induction of more efficient sample generation may alternativelybe accomplished by the inhalation of one or more of water vapour,esters, expectorant and/or menthol.

[0272] In operation, the plunger 230 is retracted so that its endsurface indicated by 270 in FIG. 4 is substantially at the second end220 of the tube 200. In such a collecting state, the tube 200 has mostof its interior surface, preferably in excess of 80% thereof, exposed tothe first end 210. Moreover, in the collecting state, a route for gasflow from the first end 210 via the gauze 250 and through the side tube240 is provided to the bag 100 (not shown in FIG. 3) or directly toambient; direct venting to ambient is preferred when, for example,screening tests for less dangerous pathogens are being undertaken.

[0273] In the collection state, the user 40 places the first end 210 tohis/her mouth so that the ring 260 engages and seals onto the user'slips. The user 40 or the tester then activates the assembly 300, forexample by depressing an associated foot pump, to eject the jet 310 ofsaline mist which the user 40 inhales. The inhaled saline mist causes anautomatic response in the user 40 to exhale forcefully causing air,mucus and/or sputum droplets in the form of a fine mist to be carriedfrom the user's 40 lungs into the tube 200. A region of the tube 200around the second end 220 forms a low velocity gas region where exhaledair from the user is inclined to flow in a vortex trajectory and therebydeposit its load of mucus and/or sputum droplets onto internalside-walls of the tube 200. Moreover, the user's 40 exhaled air isvented through the side tube 240 at a relatively high velocity.

[0274] A negative partial pressure may additionally be employed toassist in obtaining the sample.

[0275] A sample for analysis is thereby provided on the inside surfaceof the tube 200, especially in the region of the second end 220. Theplunger 230 is then actuated relative to the tube 200 for collecting thesample from the inside surface of the tube 200 and then for depositingthe collected sample onto an optical interrogation surface of theplunger 230 for subsequent optical interrogation and analysis; suchactuation also preferably includes rotation of the plunger 230 relativeto the tube 200.

[0276] The plunger 230 is thus especially adapted to collecting andmechanically concentrating the sample. Implementations of the plunger230 will now be described with references to FIGS. 4a and 4 b.

[0277] In FIGS. 4a and 4 b, the plunger 230 is substantially cylindricalin form and comprises a central hollow region 400. The plunger 230 isopen at its first end and includes a circular flange 410 for abuttingonto the second end 220 of the tube 200 when the plunger 230 is fullyinserted into the tube 200, thereby limiting an extent to which theplunger 230 can be pushed into the tube 200. The plunger 230 comprisesthe end surface 270 whose plane is substantially perpendicular to acentral longitudinal axis of the plunger 230. At an eccentric region ofthe surface 270 as shown, there is included an optically transmissiveprism 420 extending into the region 400 and a spoon-like projection 430extending outwardly from the surface 270 remotely from the region 400.The spoon-like projection 430 is susceptible in use to scoop-up mucusand/or sputum from the inside surface of the tube 200 thereonto. Thespoon-like projection 430 extends radially at the surface 270 to aperipheral extent of the surface 270. A peripheral edge 440 of theprojection 430 is preferably arranged to slidably contact onto theinterior surface of the tube 200. An optical aperture 450, also known asa prism window, is included in the end surface 270 so that the prism 420is in optical communication with sample sputum and/or mucus collectedonto the projection 430.

[0278] The projection 430 is preferably curved over towards its edgeremote from the peripheral edge 440 as shown in FIG. 3b to improveperformance of the projection 430 to retain thereon sputum and/or mucuscollected from the interior surface of the tube 200.

[0279] Moreover, the projection 430 is preferably fabricated from acompliant plastics material and arranged to be bendable to squash itsload of sample material efficiently across the optical aperture 450 whenthe plunger 230 is advanced fully into the tube 200 and is resilientlypushed against the aforementioned sealing cap (not shown); suitableplastics materials for the projection 430 include one or more ofpolyvinylchloride (PVC), nylon, polytetrafluoroethylene (PTFE),polyethylene, polypropylene, alkylene and silicone rubber. If required,the sealing cap can be provided with a recess for accommodating theprojection 430 so that a flat non-recessed end surface of the sealingcap adjacent to the recess extends to push and spread a buildup ofsample material on a side region of the projection 430 facing towardsthe optical aperture 450 substantially uniformly onto the aperture 450,thereby providing the system 10 with enhanced detection sensitivity. Ifrequired, the thickness of the projection 430 can be reduced to arelative thin neck where it joins onto the end surface 270 to provide aform of hinge so that the projection 430 is tiltable as a flap to squashsample material onto the optical aperture 450.

[0280] In order to simplify design of the projection 130 of the readerunit 50, the projection 130 adapted for insertion into the hollow region400 the plunger 230, it is desirable that the prism 420 is mountedeccentrically but away from the peripheral extent of the surface 270.When the prism 420 and its associated optical aperture 450 are arrangedin such a manner, the projection 430 is preferably of a generally“V”-shape form as illustrated in FIG. 4c. Such a “V”-shape is especiallyeffective at collecting and retaining a substantial mass of collectedsample thereon. Alternatively, as illustrated in FIG. 4b, the projection430 can be of continuously curved form; preferably, the projection 430at its remote edge is also curved towards the optical aperture 450nearer towards a central longitudinal axis of the plunger 230.

[0281] The plunger 230 is fabricated as a hollow member so that plunger230 is capable of receiving the projection 130 of the reader unit 50into the region 400. The projection 130 comprises an electronics moduleand is preferably of solid cylindrical form as illustrated in FIG. 5 andindicated generally by 500. Whereas, in use, the sample tube 200 and itsassociated plunger 230 are designed to be disposable items, the readerunit 50 is arranged to be reusable as it comprises moderately costlycomponent parts therein, for example a photomultiplier tube and a diodelaser, which will be described in more detail later. The projection 130is preferably of elongate form comprising a first end 520 and a secondend interfacing onto a module including the display 60. The first end520 comprises an eccentrically-disposed optical interfacing region 510disposed so as to align with the aperture 450, namely the prism window,when the projection 130 is inserted into the hollow region 400 of theplunger 230 to interrogate a sample collected onto the spoon-likeprojection 430.

[0282] The sample tube 200, the plunger 230 and the projection 130 areof advantage in that they are capable of being compact in storage onaccount of their mutually concentric mountability. Moreover, the tube200 in combination with the plunger 230 is capable of collecting andmechanically concentrating substantially the entire sample exhaled bythe user 40. Furthermore, the plunger 230 is capable of enabling thereader unit 50 to interrogate the sample without coming into contactwith the sample, thereby rendering the reader unit 50 reusable;operational costs are thereby reduced.

[0283] The sample tube 200 with its associated side tubes, and theplunger 230 are preferably manufactured from plastics materials, forexample one or more of an acrylate, polyethylene, polypropylene, nylon,silicone rubber, polyvinyl chloride (PVC), alkylene, polycarbonate, andpolytetrafluoroethylene (PTFE) plastics materials. More preferably, atleast one of the tube 200 and the plunger 230 are injection molded.Alternatively, one or more of the sample tube 200 and its associatedside tubes, and the plunger 230 can be fabricated from extruded metalsheet, or even fabricated by die-cast metal techniques.

[0284] Most preferably, the sample tube 200, the plunger 230 and itsassociated port 240 and filter 250 are molded as a single componentpart. Likewise, the plunger 230 with its associated projection 430 andprism 420 are preferably molded as a single component from asubstantially optically transparent plastics material, for example apolycarbonate or acrylic plastics material. Alternatively, the plunger.230 can be fabricated from a substantially black plastics material, forexample PVC, and the prism 420 subsequently assembled thereinto; the usePVC is of advantage in shielding the prism 420 from stray ambientillumination, and also shielding the remote end of the projection fromambient illumination when inserted into the plunger 230 duringmeasurement.

[0285] At the end surface, the plunger 230 can optionally include asmall orifice, for example a substantially round orifice having adiameter in a range of 0.1 mm to 2.5 mm. This orifice is of advantagefor injecting an atomized spray mist, for example a saline mist, intothe sample tube 200 when deployed to collect a sample of mucus and/orsputum from the user 40. Injection of such a mist into the tube 200prior to the user 40 exhaling the sample for collection onto innersurfaces of the tube 200 is of benefit in obtaining substantialquantities of mucus droplets from the user 40. For other types ofbioassay, it is found that the addition of a small quantity of liquid,for example a saline or a buffer solution, is desirable in that itassists the diffusion of microbes, for example bacteria, to be testedtowards the optical aperture 450. Such liquids can be added to thesystem 10 either before or after sample collection, as appropriateusing, for example, an aerosol spray or droplets from a pipette.

[0286] The tube 200, the plunger 230 and the projection 130 areadvantageously fabricated to be within preferred size ranges. Forexample, the sample tube 200 preferably has a diameter in a range of 20mm to 30 mm. Moreover, the side tube 240 preferably has a diameter in arange of 1 mm to 10 mm, more preferably in a range of 5 mm to 8 mm.Furthermore, the sample tube 200 preferably has a length in a range of40 mm to 150 mm, more preferably in a range of 50 mm to 80 mm. Theoptical aperture 450 preferably has an area in a range of 9 mm² to 64mm². It will be appreciated that these dimensions are appropriate forthe system 10 designed for use with human subjects. Other species willrequire these dimensions to be appropriately modified.

[0287] The sample tube 200 and its associated plunger 230 are preferablyprovided with a locking mechanism such that when the mucus and/or sputumsample has been mechanically concentrated within the tube 200 anddelivered efficiently onto the optical aperture 450 and the plunger 230moved to its measurement position, the plunger 230 is mechanicallylocked into position relative to the sample tube 200. Such a mechanismis of advantage in that it is capable of preventing the sample tube 200and its plunger 230 being reused; in poorer parts of the world, there isa temptation to reuse medical parts, for example syringes. Morepreferably, insertion of the projection 130 of the reader unit 50triggers engagement of such a mechanism to prevent reuse. A temptationfor reuse can potentially occur where the system 10 displays anon-positive indication for the presence of a pathogen. Such a lockingmechanism is of further advantage in that locking of the sample tube 200to the plunger 230 together with the sealing cap forms an enclosedregion for isolating dangerous pathogens. Yet more preferably, thesample cap also snap engages onto the tube 200 so that the two cannotsubsequently be disengaged.

[0288] In order to enhance a vortex generated within the sample tube 70,200 and thereby improve sputum and/or mucus deposition therein, the tube70, 200 can be provided with additional features to modify air flowtherein. Referring to FIG. 6, there is shown a modified samplecollection tube indicated generally by 600, the tube 600 including anannular septum orifice 610 included between the atomizer assembly 300and the side tube 240. The septum 610 is preferably included as near aspossible to the end 210. Moreover, the septum orifice 610 is preferablemolded to be an integral part of a cylindrical part 620 of the tube 600.The projection 430 of the plunger 230 is preferably made flexible sothat advancing the plunger 230 into the tube 600 after sample collectiontherein causes the projection 430 to flex against the septum orifice 610to squash the sample onto the optical aperture 450.

[0289] The septum orifice 610 assists by enhancing peripheral drag togenerate eddies and corresponding complex multiple vortex formation,thereby enhancing mucus and/or sputum deposition on an inner surface ofthe cylindrical part of the tube 600. Moreover, the orifice 60 alsoassists to prevent saline mist encroaching into regions of the tube 600remoter from the user 40 in use. Preferably, the septum orifice 610 isrecessed behind the end 210 by a distance in the order of 5 mm to 15 mm.Moreover, the septum orifice 610 preferably includes a central holehaving a diameter in a range of substantially 3 mm to 20 mm. The septumorifice 610 is preferably fabricated from a collapsible material (forexample a flexible plastic) in order that the sample collected on thewalls of the sample tube 200, closest to the user's 40 mouth, can beconcentrated onto the optical aperture 450.

[0290] The sample tube 200 can alternative be modified to include adirectional bend near the opening 210 to generate a sample collectiontube indicated generally by 650. The assembly 300 is preferably includedon an outside bend portion of the tube 650 as illustrated to injectsaline mist towards the end 210. The directional bend causes anasymmetrical spatially varying drag to air that promotes eddy formation.

[0291] In order to obtain superlative collection performance, acombination of the features of the tubes 600, 650 can be employed.

[0292] Further alternative embodiments of sample collection unit 30 arepossible.

[0293] For example, in FIG. 7, there is shown a sample collectionchamber indicated generally by 700. The chamber 700 comprises an inletpipe 720 of substantially 25 mm diameter for delivering exhaled breathfrom the user 40 to a collection box 710. More preferably, the inletpipe 720 has a diameter in a range of 18 mm to 30 mm. The collection box710 includes at its periphery a prism 750 susceptible to promotingevanescent radiation propagation at an exposed surface thereof facinginwardly into the box 710 where mucus and/or sputum deposition occursfrom the exhaled breath. Sample collection onto the exposed surface ispromoted by a vortex generated within the box 710; this vortex isespecially enhanced when an exit pipe 760 from the box 710 has adiameter which is less than that of the inlet pipe 720. Preferably, thediameter of the exit pipe 760 is substantially 5 mm, although a diameterin a range of 2 mm to 10 mm is especially preferred. The prism 750 canbe situated in any position on the walls of the box 710 in order tocollect samples thereon.

[0294] Exhaled breath from the box 710 is beneficially conveyed alongthe exit pipe 760 to the bag 100 and its associated gas exit orifice105. Alternatively, the exhaled breath output from the box 710 can beinitially passed through a filter to remove pathogens and then vented toambient or to the bag 100. Beneficially, a venturi can be incorporatedinto the inlet and/or exit pipes to assist with vortex formation withinthe box 710. The exit pipe 760 and inlet pipe 720 can be placed at anyposition relative to each other and the prism 750, within the box 710.

[0295] The projection 130 of the reader unit 50 will now be furtherdescribed in more detail with reference to FIGS. 8a and 8 b. In order toobtain optimum readout from the prism 420, optical interrogationcomponents of the reader unit 50 are preferably housed within theassembly. The projection 130 therefore comprises a photomultiplier tube(PM tube) 800 and a diode laser. Advantageously, the PM tube 800 is aproprietary device manufactured by Hamamatsu Photonics K.K. of Japan,the device having a part number R7400U-01. A photosensitive face of thePM tube 800 is orientated towards the optical interface region 510,preferably spatially as close thereto as possible. Moreover, theprojection 130 further comprises a solid state diode laser 810. Aconfiguration depicted in FIG. 8a is most preferred as it results inleast optical losses when coupling optical radiation to the opticalaperture 450. However, especially when the projection 130 is of arelatively small exterior diameter, it is convenient for the diode laser810 to be coupled via a light guide 820, for example comprising aparallel bundle of optical fibre waveguides. In a standard relativelylarger version of the projection 130 illustrated in FIG. 8b, the diodelaser 810 and the PM tube 800 are mounted mutually adjacently, therebyrequiring only a relatively shorter length of light guide to beemployed. However, in a compact relatively smaller version of theprojection 130 depicted in FIG. 8b, the diode laser 810 is positionedbehind the PM tube 800 as illustrated and a relatively longer section oflight pipe 830 employed to convey light from the diode laser 810 to theinterface region 510. If required, the projection 130 can be fabricatedfrom diecast, machined or extruded metal and the diode laser 810 can bethermally coupled to the peripheral wall of the projection 130 forcooling purposes. Similar thermal considerations pertain to the PM tube800 although this device dissipates relatively negligible power inoperation. Design of the reader unit 50 comprising the projection 130and its associated parts will be described in more detail later.

[0296] Referring again to FIG. 2, the aforementioned sealing cap appliedto the collection tube 70 is denoted by 900. The cap 900 is illustratedin cross-section in more detail in FIG. 9, the cap 900 comprising a mainbody component 905, first and second liquid reservoirs 910, 920including liquid masses 930, 940 respectively, and a sealing top 950hermetically bonded to the body component 905 to seal the liquid masses930, 940 into the cap 900. The body component 905 and the sealing top950 are preferably fabricated from a highly flexible plastics material,for example soft silicone rubber. The sealing top 950 is substantially arelative thin flexible membrane including domed regions 960, 970 alignedto corresponding reservoirs 920, 910 respectively. Bonded centrally tothe domed regions 960, 970 are steel pins 980, 990 respectively. Thesteel pins 980, 990 have blunted broadened ends where they are moldedinto the sealing top 950, and sharp pointed ends where they face towardsend regions of the reservoirs 920, 910. The sealing cap 900 furthercomprises a retaining feature 1000, for example a barbed insert withbackwardly directed barbs which bind into the collection tube 70, 200 toallow the cap 900 to be easily inserted into the tube 70, 200 but notremoved therefrom again.

[0297] In operation, the user 40, or preferably the tester, depressesthe domed regions 960, 970 to cause the pins 980, 990 to puncture theirrespective reservoirs 920, 910 to release the contents of theirrespective liquid masses 940, 930 into the sample tube 70, 200 tochemically process mucus and/or sputum collected at the optical aperture450 of the plunger 110, 230.

[0298] In manufacture, the body component 905 is orientated so that itsend face 1010 is downwardly facing. The reservoirs 910, 920 are thenfilled with their respective liquid masses 930, 940 and then the sealingtop 950 comprising its pins 980, 990 is ultrasonically welded, orotherwise hermetically bonded, into a recess molded into the bodycomponent 905 as illustrated.

[0299] Although two reservoirs 910, 920 are shown in FIG. 9, it will beappreciated that the sealing cap 900 can be fabricated to have one ormore reservoirs. Moreover, the liquid masses 930, 940 can be varied incomposition depending upon the type of pathogen to be detected by thesystem 10. For example, one of the liquid masses can be a biologicalbacterial lysing agent whereas another of the liquid masses can be abiological fluorescent marker agent. These agents will be described inmore detail later.

[0300] 3.2 Reader Unit

[0301] The reader unit 50 illustrated in FIG. 1 will now be described inmore detail.

[0302] Referring to FIG. 10, there is shown an optical configurationindicated generally by 1100. This configuration 1100 is employed in thesystem 10 and its parts are distributed between the reader unit 50 andits associated projection 130, and the plunger 110, 130.

[0303] In particular, the reader unit 50 in its projection 130 includesthe laser 810, the light guide 820, 830 (if required), an optical filter1120, the PM tube 800 (and associated power supply, not shown) and aminiature solid-state diode detector 1130. A main part of the readerunit 50 includes the display 60, a microcontroller 1150 for executingcalculations, a synchronous demodulator 1140, and a strobe circuit 1160.

[0304] The plunger 110, 230 comprises a prism 420; in particular, thepreferred prism 420 is a dove-type prism of a cross-sectionaltrapezoidal form as illustrated. A major front face of the prismprovides the optical aperture 450 that is coated in a biologicallyactive layer 1110 which will be described in further detail later.

[0305] Component part interconnection within the configuration will nowbe described with reference to FIG. 10. The microcontroller 1150includes a data output which is coupled to the display 60. In itssimplest configuration, the display 60 merely includes a yes/noindicator for indicating whether or not a given pathogen is present inthe mucus and/or sputum sample above a predefined threshold. In a morecomplex configuration, the display 60 provides a quantitative measure ofthe concentration of pathogens present in the mucus and/or sputumsamples being interrogated; the display 60 can be one or more of aliquid crystal display (LCD), for example an alpha-numerical LCDdisplay, a light emitting display (LED) and a miniature plasma display.The microcontroller 1150 is also connected at its output to the strobecircuit 1160 for modulating power applied to the laser 810, therebytemporally modulating its optical output beam 1200 launched into thelight guide 820, 830. Moreover, the microcontroller 1150 also includesan input S₃ for receiving a demodulated output signal from thesynchronous demodulator 1140. The PM tube 800 includes a signal outputS₁ which is connected to a signal input of the synchronous demodulator1140. Furthermore, the diode detector 1130 includes a signal output S₂which is coupled to a strobe input of the demodulator 1140. The opticalfilter 1120 is included between the PM tube 800 and a smaller majorrear-plane face 1170 of the prism 420 as illustrated; the filter 1120 iseffective at transmitting radiation components arising from evanescentwave interaction in the active layer 1110 and reflecting and/orabsorbing scattered radiation generated directly from scatter of primaryradiation within the prism 420.

[0306] Operation of the optical configuration will now be described withreference to FIGS. 1 and 10. The user 40 activates the system 10 whichcauses the microcontroller 1150 in combination with the strobe circuit1160 to generate a modulated signal to drive the diode laser 810 andthereby generate the correspondingly strobed beam 1200. Preferably, thewavelength of radiation output from the laser 810 will be selected tomatch the excitation wavelength of fluorophores employed to analyse themucus and/or sputum sample collected onto the optical aperture 450. Suchfluorophores can be selected to be selectively responsive at differentwavelengths, for example deep red, green, or blue radiation wavelengths.The use of longer wavelength radiation from the laser 810 correspondingto red radiation is preferable to reduce costs as red lasers are highlyinexpensive and readily available. Conversely, solid state laser diodescapable of outputting radiation at relatively shorter blue and greenradiation wavelengths are presently relatively expensive. However, a PMtube that is sensitive in the red spectral region must be used and theseare slightly more expensive than those most sensitive in the blue/greenregion. However, the optical configuration 1100 is, therefore, capableof being operated at different wavelengths to match the type offluorophores employed to analyse the mucus and/or sputum sample, and thesystem 10 can be constructed to work at any optical frequency desired.

[0307] The beam 1200 is preferably strobed at a frequency in a range of100 Hz to 100 kHz. More preferably, the beam 1200 is strobed at afrequency in a range of 100 Hz to 1500 Hz. Most preferably, the beam1200 is strobed at a frequency of substantially 1030 Hz as this rendersamplifier circuits (not shown) associated with the PM tube 800 and thesynchronous demodulator 1140 straightforward to design using standardcomponents as bandwidth constraints are not especially problematic atsuch a strobe frequency. Moreover, 1030 Hz is not a harmonic of 50 Hzmains supply, thereby rendering the system 10 less susceptible to beaffected by 50 Hz fluctuating light sources such as mains-operatedfluorescent strip lights frequently found in hospitals and clinics.

[0308] Preferably, the strobe circuit 1160 is operable to modulate theinjection current used to excite the laser 810 so that this current isperiodically switched above and below the lasing current threshold ofthe laser 810. Alternatively, the laser 810 can be operated at constantoutput intensity and a separate modulator device, for example a liquidcrystal (LCD) cell, used to temporally modulate an output beam from thelaser 810 to generate the radiation beam 1200.

[0309] The laser 810 emits the strobed beam 1200 which propagatesthrough the light guide 820, 830 to generate a corresponding exit beam1210 which propagates to a first inclined face 1215 of the prism 420 andis refracted thereat to generate a corresponding refracted beam 1220which subtends an angle θ relative to a normal to the plane of theoptical aperture 450. Preferably, the angle θ is in a range of 62° to80°. More preferably, the angle θ is substantially 70°.

[0310] The refracted beam 1220 propagates to the optical aperture 450and is mostly reflected thereat to generate a reflected beam 1230 thatthen propagates to a second inclined face 1235 of the prism 420 toemerge refracted therefrom as a beam 1240 which then propagates to thedetector 1130. The beam 1240 gives rise to a strobe signal at the outputS₂ which is used as a modulation reference signal for the demodulator1140.

[0311] Where the beam 1220 impinges onto the optical aperture 450, afraction of the radiation present in the beam 1220 is coupled into theplane of the aperture 450 in the form of an evanescent wave 1245. Thisevanescent wave 1245 propagates in a boundary region at the interface ofthe biologically active layer 1110 to the prism 420 itself. The boundaryregion is frequency dependant and at the frequencies in which the systemworks this is effectively only in the order of 100-200 nm thick. Thus,coupling of the beam 1220 to form the evanescent wave 1245 allows forextremely efficient optical interrogation of chemicals present at theboundary region. If fluorophores are present at the boundary region,they are excited by the evanescent wave to generate fluorescentradiation. Preferably, this fluorescent radiation is at a differentradiation frequency to that of the beam so that the filter 1120 can beused to discriminate scattered radiation from the beam 1220 fromfluorescence at the aforementioned boundary region; namely, fluorophorespresent at the boundary region are operable to provide radiationwavelength conversion.

[0312] Fluorescent radiation generated at the boundary region propagatesfrom the boundary region through the prism 420 to exit from the prismface 1170 and propagate through the filter 1120 to the PM tube 800 tocause a corresponding sense signal to be generated at the output S₁. Thesense signal passes to the signal input of the demodulator 1140 and issynchronously demodulated therein with respect to the signal from theoutput S₂ to provide a demodulated signal at the output S₃ which passesto the microcontroller 1150 for subsequent sampling and conversion tocorresponding data D. The microcontroller 1150 then proceeds to comparethe data D with a preprogrammed threshold level T and determine therebywhether or not pathogens are present in the mucus and/or sputum samplescollected onto the optical aperture 450 and interrogated by theevanescent wave radiation propagating therealong.

[0313] Preferably, the degree fluorescence, namely the magnitude of thedata D, can be determined prior to, namely providing data D1, and thenagain after, namely providing data D2, collecting and mechanicallyconcentrating the sample of sputum and/or mucus onto the opticalaperture 450. A difference value given by Equation 1 (Eq.1):

ΔD=modulus (D ₂ −D ₁) Eq. 1

[0314] is then calculated in the microcontroller 1140. This differencevalue AD is then compared with the threshold value T to determinewhether or not pathogens are present in the sample. Such a differencemethod of measurement is effective at removing systematic contributionsto the sense signal provided at the output S₁; such systematiccontributions can arise from scatter within the prism 420, residualfluorescence within the prism 420 especially if it is fabricated fromplastics materials, and finite radiation wavelength discriminationprovided by the filter 1120. Suitable plastics materials for fabricatingthe prism 420 include perspex, acrylate, polycarbonate andpolymethylmethacrylate (PMMA). It should be noted that the use ofpolymer materials that exhibit fluorescence is be avoided wherepossible.

[0315] Preferably the threshold value T is made proportional to theoptical interrogation radiation power delivered into the beam 1210 fromthe laser 810. More preferably, the threshold value T is madeproportional to the radiation power in the beam 1240 received at thedetector 1130 so as to account for efficiency of optical coupling intothe prism 420 which can potentially vary from plunger 230 to plunger230, especially if mechanical tolerances in manufacture are not tightlycontrolled.

[0316] Although the use of the PM tube 800 is described in theforegoing, it will be appreciated that other types of optical detectorscan potentially be employed, for example avalanche photodiodes,phototransistors or low-noise photodiodes. If signal-to-noiseconsiderations allow, the laser 810 is preferably substituted with alower-cost high-brightness light emitting diode (LED).

[0317] If required, the filter 1120 can comprise several optical filtercomponents to enhance its wavelength discrimination, for example byutilizing several diffraction grating layers. Moreover, themicrocomputer 1150 can be programmed to account for systematic steadytemporal drift in the sense signal to account for warm-upcharacteristics of the system 10 when activated from a cold state. Anestimate of such drift can be made by interrogating the optical aperture450 for a period of a few minutes before introducing the mechanicallyconcentrated sample thereto.

[0318] Preferably, the microcontroller 1150 includes a data logger forrecording test results and corresponding reference codes for subsequentdownloading to a database from the reader unit 50. In such aconfiguration, the reader unit 50 preferably includes a data entry keypad so that each test performed by the reader unit 50 can be allocatedan identification reference. When the microcontroller 1150 is configuredto provide a data logging characteristic, the microcontroller 1150 canpotentially be used effectively during a disease epidemic to generatepathogen infection rate statistics.

[0319] It will be appreciated that the system 10 can be adapted forinterrogating liquid samples from other sources than exhaled breath.Referring to FIG. 11, there is shown a alternative configuration forpart of the system 10 for analysing a liquid, for example a bloodsample, flowing or stagnant within a tube 1310. The prism 420 is anintegral part of, or is attached to, a side region of the tube 1310. Thebeam 1210 passes through the prism 420 as the beams 1220, 1230 andexcites fluorophores attached to the major face of the prism 420 facingin contact towards the liquid sample within the tube 1310. Fluorescenceof the fluorophores in response to composition of liquid sample, forexample blood, is received by the PM tube 800 to generate a strobe sensesignal at the output S₁ for subsequent synchronous detection. The system10 modified according to FIG. 11 is thus susceptible to providecontinuous monitoring of blood or other body fluids, for example urine,for pathogens and therefore has widespread potential application inhospitals and body fluid processing facilities.

[0320] Moreover, if required, a sample air stream can be continuouslypassed through the tube 1310 to detect for air-borne pathogens, toxicpollutants and explosive vapours for example. Thus, the biologicalmeasurement system 10 is potentially adaptable to other applicationsother than merely to detect respiratory pathogens such as tuberculosis.

[0321] The dove-type prism 420 can be substituted in the plunger 110,230 with an alternative prism indicated generally by 1400 in FIG. 12.The prism 1400 preferably has internal angles of 55°, 125°, 70° and 110°as illustrated. Optionally, a face indicated by 1410 can be a mirrorcoated surface to enhance reflective performance of the face 1410 whenreflecting the beam 1210 to form the beam 1220. By reducing internalreflection losses, the prism 1400 is potentially capable of imparting anenhanced detection signal-to-noise ratio to the system 10.

[0322] Furthermore, dove-type or other prisms where the angle ofacceptance is such that multiple reflections are induced within theprism can be adopted. For example, suitable alternative prisms aredescribed in a book by C. N. Banwell and E. M. McCash, “Fundamentals ofMolecular Spectroscopy” (1994) McGraw-Hill, 4^(th) edition which isherewith incorporated by reference.

[0323] It should be appreciated that fluorimetry has been shown to be ofconsiderable importance for the detection of biological materials suchas proteins and DNA, where fluorophores on antibodies are used asmarkers for detection. Detection using such fluorimetry can be executedby way of either

[0324] (a) bulk fluorescence, measurements; or

[0325] (b) through the application of interrogation techniques such asevanescent wave detection; or

[0326] c) cavity ring down spectroscopy; or

[0327] (d) through the use of displacement assays.

[0328] Such fluorimetry offers some potential advantages in terms ofspecificity, simplicity, and sensitive. Evanescent wave detection iswell known, but low-cost evanescent wave fluorimeters are not yetcommercially available for use in pathogen detection as described withrespect to the present invention.

[0329] It should be further appreciated that sample analysis followingcollection onto an optical interrogation area can be undertaken usingmeans other than fluorimetry, for example, radioactive andphosphorescent markers can be utilized or chemiluminescence techniques.

[0330] Detection can also be carried out using other forms ofspectroscopy, not associated with immunoassay systems or evanescentwaves. For example, infrared spectroscopic methods can identify thepresence of specific molecular fragments on the basis of ‘groupfrequencies’ at specific regions of the infrared spectrum; these can beperformed using both transmission and reflection geometries where thelatter detects the absorption of evanescent IR radiation. Otherpossibilities are preferably, but not exclusively, Surface Acoustic Wave(SAW) detection and Surface Plasmon Resonance (SPR) which may providesignal enhancement and thus gains in sensitivity.

[0331] 4. System Biochemistry

[0332] In the foregoing, the measurement system 10 is described withrespect to its hardware. In the following description, chemical aspectsof the system 10 will now be elucidated in more detail.

[0333] 4.1 Biochemical Overview

[0334] The measurement system 10 is capable of operating according totwo alternative detection methods, namely either:

[0335] (a) by fluorophore displacement resulting from the presence of apathogen (namely competitive displacement assay); or

[0336] (b) by fluorophore binding promoted by the presence of a pathogen(namely selective binding assay).

[0337] In the competitive displacement assay, the sense signal at theoutput S₁ reduces as the pathogen is introduced into the samplecollection unit 30. Conversely, in the selective binding assay, thesense signal at the output S₁ increases as the pathogen is introducedinto the collection unit 30. Both assays are pertinent as certain typesof pathogen are best detected by one or other of the assays.

[0338] In the selective binding assay, the optical aperture 450 iscoated during manufacture with a first antibody that will bind to thepathogen to be detected. In operation, the pathogen is mechanicallyconcentrated onto the optical aperture 450 and becomes immobilizedthereat on account of its affinity to the first antibody. Next,fluorophores bound to second antibodies having affinity to the pathogenare released into the sample tube 70, 200 so that the fluorophoresbecome bound to the pathogen immobilized to the first antibodies at theoptical aperture 450. If required, the first and second antibodies canbe identical although this is not essential as pathogens frequentlyexhibit several surface regions to which different antibodies can bind.The second antibodies and their associated fluorophores can be in theform of a liquid held in one of the reservoirs 910, 920 of the sealingcap 900. When the pathogen has been immobilized directly to the firstantibodies at the optical aperture 450 together with the fluorophoresbound to the second antibodies immobilized to the pathogen, theevanescent wave radiation 1245 is capable of interacting strongly withthe fluorophores, thereby generating significant fluorescence fordetection by the PM tube 800.

[0339] In the competitive binding assay, the optical aperture 450 iscoated during manufacture with the first antibody that will bind thepathogen being investigated. Moreover, during manufacture, fluorophoresbound to analogues of the pathogen that bind weakly to the firstantibody are added to the optical aperture 450. When the mechanicallyconcentrated sample is applied to the aperture 450, the pathogen thereindisplaces the weakly bound fluorophores and associated analogues andbind in substitution to the immobilized first antibodies. The weaklybound fluorophores and associated analogues, when displaced, migrateaway from the boundary region supporting the evanescent wave propagation1245 causing a decrease in fluorescence detected by the PM tube 800.Preferably, one or more of the reservoirs 910, 920 of the sealing cap900 includes a wash agent to assist removal of the displacedfluorophores bound to associated analogues from the optical aperture 450so that a final settled reading is more rapidly attained.

[0340] It should be noted that the antibodies that can be used for thesetests may be monoclonal or polyclonal in form.

[0341] 4.2 Antibody Immobilisation

[0342] The immobilisation of antibodies to glass or plastic surface, forexample to the optical aperture 450, is already well studied, and manyprotocols exist. These protocols derive largely from the success ofknown ELISA tests, in which antibodies immobilised on a 96-well plateform a crucial component of such tests. In a textbook “mobilizedBiomolecules in Analysis: a practical approach”, edited by T. Cass andF. S. Ligler (Oxford University Press), said textbook herewithincorporated by reference, there is provided a thorough overview of manyimmobilisation protocols.

[0343] Such protocols will each typically comprise:

[0344] (a) a preparation step, in which a surface is cleaned andoptionally activated;

[0345] (b) an incubation step during which antibodies or antibodyfragments are attached to the surface; and then

[0346] (c) a blocking step to prevent further non-specific binding ofbiomolecules to the surface

[0347] Rinsing steps generally follow the incubation and blocking steps.The prepared surface is then dried and stored in a dry atmosphere.Incubation times depend to a significant extent on the molecule (namelypathogen) to be captured and the surface composition.

[0348] Activation of the surface is commonly achieved by irradiating thesurface, or exposing it to plasmas or chemicals such as silanesincorporating an active group to which antibodies can be bound. Oncechemically active groups exist on the surface, a simple incubation stepis usually sufficient to bind the antibodies, such antibodies alsoreferred to in the following as receptors.

[0349] 4.3 Analogues for the Competitive Binding Assay

[0350] Analogues used in the aforementioned competitive displacementassay correspond to molecules or molecular groups which bind toreceptors, for example antibodies, immobilised on the optical aperture450 of the prism 420, but do so with a lower association constant thanthe pathogen to be detected. Preferably, the analogue-receptorassociation constant is less than 10% of the pathogen-receptorassociation constant, and if possible less than 1%.

[0351] These analogues may be similar molecules from closely relatedspecies, for example sheep luteinising hormone is capable of providingan analogue of human chorionic gonadotropin. Alternatively, theanalogues can be molecules synthesised to mimic the structure of thepathogen, especially the epitope to which the antibody binds, or amodified version of the pathogen.

[0352] It is also potentially advantageous to use derivatives of thepathogen as analogues. Such derivatives can be artificial derivativessuch as molecules modified by adding bulky or ionic groups (which canreduce the binding energy), by adding steric or charge interference, orbinding to a bulky group, which can cause a conformational change in thebinding site of the pathogen. In the case of analytes which areproteins, the corresponding amino acid sequence of the protein can bemodified near the binding site to the receptor by recombinant molecularbiology techniques, such as site directed mutagenesis. Alternativelythey can be natural metabolites of the target analyte.

[0353] Techniques required to prepare such analogues are known, andthere is much prior art on the design of such analogues. Chemicalmodifications of organic molecules, biochemical modifications ofnaturally occurring molecules and synthesising structural mimics ormolecules are known in the art. The suitability of a candidate analoguecan be determined by a competitive ELISA assay between the analyte andthe candidate analogue or by a measurement of the association constantwith the receptor.

[0354] Polyclonal and monoclonal antibodies reactive against pathogenssuch as mycobacterium tuberculosis are readily available from severalcommercial vendors, for example Skybio Ltd. in the United Kingdom. Suchantibodies can be purchased in sizeable batches and labeled andimmobilized using standard known chemistries.

[0355] 4.4 Fluorophores

[0356] Selection of suitable fluorophores for use in the system 10 hasan important bearing on the technical performance of the system 10, forexample its signal-to-noise ratio and hence its ability to identifyearly onset of disease.

[0357] There are many commercially available fluorophores. The mostsignificant qualities of such fluorophores are:

[0358] (a) their absorption band, which limits the range ofinterrogating radiation wavelengths at which they can be excited; and

[0359] (b) their emission band, namely the range of wavelengths overwhich fluorescent radiation is emitted from the fluorophores whenexcited.

[0360] The absorption band must overlap as much as possible with thespectrum of the interrogating light source used, namely the laser 810 inthe system 10; moreover, the emission band should overlap as little aspossible with the absorption band. These qualities limit the range offluorophores which can be used in the system 10. Other factors which mayinfluence the choice of an optimal fluorophore for the system 10 are:

[0361] (a) the ease with which the fluorophore can be coupled to acorresponding target molecule, for example an antibody or analogue; and

[0362] (b) the separation between the absorption and emission bands ofthe fluorophore; and

[0363] (c) the brightness of the fluorescent radiation emitted from thefluorophore.

[0364] A commercial company Molecular Probes manufactures and supplies acommercial range of fluorescent dyes called the Alexa dye series. Thisseries includes a number of bright fluorophores with optimal excitationwavelengths ranging from 346 nm to 684 nm, including many moleculesspecifically designed to work well with common light sources such asbright laser diodes or red LEDs. Many other dyes exist and are widelyused, for example fluorescein isothiocyanate (FITC), BODIPY,phycoerythrin, allophycocyanin (APC), rhodamine, Texas Red and OregonGreen. Some of these dyes and their relevant parameters are listed inTable 1; one or more these dyes can, if required, be employed in thesystem 10 either alone or in combination. TABLE 1 Examples of typicalfluorophores susceptible for use in the system 10. Absorption Emissionpeak peak Dye name Abbreviation (nm) (nm) Fluorescein isothiocyanateFITC 493 520 R-phycoerythrin RPE 495, 536 576 B-phycoerythrin BPE 546576 Rhodamine — 550 573 Rhodamine B — 578 604 Allophycocyanin APC 630,645 655, 660 Alexa Fluor 350 — 346 442 Alexa Fluor 430 — 433 539 AlexaFluor 488 — 495 519 Alexa Fluor 532 — 532 554 Alexa Fluor 594 — 590 617Alexa Fluor 633 — 632 647 Alexa Fluor 680 — 684 707 BODIPY 493/503 — 500506 BODIPY 665/676 — 665 676 Cy5 — 649 666, 670 Texas Red — 595 620Teramethyl rhodamine TRITC 550 573 isothiocyanate

[0365] In order to enhance performance, latex spheres includingfluorescent materials can be bound to one or more of antibodies andanalogues in order to provide the system 10 with enhanced detectionsensitivity. Such latex spheres are capable of exhibiting an enhanceddegree of fluorescent radiation in response to being excited by theevanescent radiation wave 1245 at the optical aperture 450.

[0366] Latex spheres are commercially available from companies such asDynal Biotech with a wide range of surface chemistries; such surfacechemistries can include fluorophores and also impart the spheres withmagnetic properties. In the sample tube 70, 200, magnetic attraction oflatex spheres comprising fluorophores when mechanically concentratingthe sputum and/or mucus sample onto the projection 430 is highlyadvantageous to achieving enhanced measurement sensitivity from thesystem 10. The latex spheres used for this system are preferably in therange of 50 nm to 1 μm in diameter; more preferably, the spheres are inthe range of 100 nm to 200 nm in diameter, namely in line with theboundary depth of the evanescent wave penetration at the opticalaperture 450.

[0367] In the selective binding assay, magnetically labeled fluorescentlatex spheres can be released from one of the reservoirs 910, 920 of thesealing cap 900 into the sample tube 70, 200 prior to mechanicalconcentration of the sample at the optical aperture 450; preferably, theaperture 450 and/or the projection 430 are provided with one or more,small, movable, permanent magnet(s) thereat to assist latex spherecollection. Following collection and concentration of the sample ontothe optical aperture 450, the magnet(s) can be moved away to allow thespheres that have not been chemically bound to the aperture 450 todiffuse away into the bulk liquid, leaving only the bound species to bedetected at the aperture 450.

[0368] 4.5 Optional Lysis Sensitivity Enhancement

[0369] Sensitivity of the system 10 to the detection of pathogens can beenhanced by employing a process known as lysis. Lysis is the process ofbreaking cells, for example pathogen microbes, into its componentfragments. Antibody-labeled fluorophores are capable of binding to thesefragments. Moreover, the fragments are susceptible to binding toantibodies at the optical aperture 450.

[0370] There are a wide variety of methods used to lyse microbes, forexample bacteria. Such methods comprise one or more of chemical,mechanical and thermal processes. These processes are known to theskilled addressee. Lysis of pathogens collected within the sample tube70, 200 is of advantage in that lysis fragments are susceptible tobinding to first antibodies immobilized at the optical aperture 450 andalso to second antibodies bound to associated fluorophores. Thus, lysisis capable of enhancing the detection efficiency in the aforesaidselective binding assay within the system 10, for example by at least anorder of magnitude. Likewise, lysis is capable of giving rise to morecompetitive displacement sites at the optical aperture 450 in the caseof the aforesaid competitive displacement assay.

[0371] Chemical methods of lysis involve the use of enzymes such aslysozyme, or detergents such as SDS to break down cell walls. Mechanicalmethods physically break down cell membranes; examples of mechanicalmethods include nitrogen cavitation bombs, french press or hughes press,sonication, glass beads or osmotic lysis techniques. Thermal lysisemploys extremes of temperature excursions to destroy cell walls; suchtemperature excursions can comprise repeated freezing and thawing of acell culture.

[0372] Mycobacteria, for example mycobacterium tuberculosis, areespecially difficult to lyse. Lysis buffers specifically adapted formycobacteria comprise additional reagents such as lysozyme to break downmycobacteria cell walls.

[0373] During lysis, enzymes released from cell interior regions oftenattack molecules of interest for detection purposes within the system10. However, lysis buffers can be formulated to include additionalingredients such as protease inhibitors to prevent the target moleculefrom being digested or denatured. Table 2 provides a list of lysisprotocols susceptible for use within the system 10 to enhance itspathogen detection performance. TABLE 2 Examples of lysis protocolsPrinciples Reference Method used Class of target Gen-Probe packageinsert Sonicate for 15 minutes in Mechanical, Mycobacterium lysis bufferand glass beads chemical Pierre et al., J. Clin. Micro. 15 minutes at95° C. with Thermal, Mycobacterium 29 (4): 712-717 (1991) 0.1M NaOH, 2MNaCl, chemical 0.5% SDS Hurley et al, Int. J. 3 minutes in minibeadMechanical Mycobacterium Systematic Bacteriology 38 beater withdistilled phenol (2): 143-146 (1988) and zirconium beads Robson et al.,U.S. Pat. No. Heating for 2 to 15 minutes Thermal Mycobacterium 5376527at 60° C. to 100° C. Pierce product information Shake sample with B-PERChemical Bacterium Bacterial Protein Extraction Reagent for 10 minutes

[0374] Lysis is preferably performed in the collection apparatus 30either before mechanical concentration of the sample has occurredtherein or after mechanical concentration has been achieved.

[0375] It will be appreciated that sensitivity of the system 10 can befurther enhanced by utilizing many known amplification techniquesemployed in standard immunoassay. Such amplification techniques include,but are not limited to, biotin/axidine or biotin/streptavidin sandwichtechniques and enzyme-linked assays. Also, chromogenic substances can beused in substitution, or in addition to, the fluorophores, for exampleas in ELISA assays, producing a colour change in solution rather thanfluorescent signal as in the system 10 described above. Such colourchange can be detected electronically using colour sensitive electronicdetectors or using the naked eye.

[0376] 4.6 Description of Biochemical Interactions Within the System

[0377] In order to more completely describe operation of the system 10,especially with regard to biochemical reactions occurring therein,reference will be made to FIGS. 13 to 15.

[0378] 4.6.1 Selective Binding Assay

[0379] Referring to FIG. 13, there is illustrated a binding processwhich occurs in operation within the sample tube 200 at the opticalaperture 450.

[0380] In STEP A, the first antibodies denoted by 1500 are bound to theoptical aperture surface 450, the first antibodies 1500 deposited duringfabrication of the plunger 110, 230. The optical surface is opticallyinterrogated with evanescent wave radiation and a first degree offluorescence measured.

[0381] In STEP B, the mucus and/or sputum sample is mechanicallyconcentrated within the sample tube 70, 200 as described in theforegoing and deposited at the optical aperture 450 whereat specificpathogens 1520 of interest bind to the first antibodies 1500 asillustrated.

[0382] In STEP C, the fluorophore-labeled second antibodies 1530, 1540are released from one or more of the reservoirs 910, 920 of the sealingcap 900 by rupturing them as described earlier with reference to FIG. 9;the fluorophore-labeled antibodies 1530, 1540 wash onto the opticalsurface 450 as in STEP B and bind to the specific pathogens 1520 in STEPC. The optical aperture 450 with its bound first and second antibodies,1500, 1530, pathogen 1520 and fluorophores 1540 can then be opticallyinterrogated using evanescent wave radiation of identical magnitude asused to determine the first degree of fluorescence, thereby enabling asecond degree of fluorescence to be measured. A difference between thefirst and second degree of fluorescence gives an indication of thepresence of the fluorophores 1540 from which can be inferred thepresence of the pathogen 1520.

[0383] A variation on the process of FIG. 13 is possible as depicted inFIG. 14.

[0384] In STEP 1 of FIG. 14, the pathogen 1520 in the form of mucusand/or sputum is deposited onto inside walls of the sample tube 70, 200.

[0385] In STEP 2, the second antibodies 1530 and their associatedfluorophores 1540 in the form of latex spheres are then released fromone or more of the reservoirs 910, 920 in the sealing cap 900. Thesecond antibodies 1530 bind to the pathogen 1520 within the sample tube200. The plunger 230 is then used to mechanically concentrate thepathogens 1520 bound to their second antibodies 1530 and associatedrelatively large latex spheres. Such an order of steps means that thereis a relatively large fluid mass to collect than in FIG. 13; thisrelatively larger mass is of benefit where relatively little mucusand/or sputum is deposited within the tube 200.

[0386] In STEP 3, the pathogens 1520 bound to the second antibodies 1530and their latex sphere laden fluorophores 1540 are then presented to theoptical aperture 450 whereat they bind to the first antibodies 1500immobilized onto the aperture 450. The pathogens 1520, the antibodies1500, 1530 and the fluorophore-laden latex spheres thereby become boundto the aperture 450 and fluorescence when interrogated with evanescentradiation to signal presence of the pathogen 1520 in the sample.

[0387] 4.6.2 Competitive Binding Assay

[0388] The competitive binding assay is depicted in FIG. 15.

[0389] In STEP 1, the optical aperture 450 has bound thereto duringfabrication the first antibodies 1500. Moreover, analogues 1600 of thepathogen 1520 to be detected are added to the aperture 450 for weaklybinding to the immobilized first antibodies 1500. The analogues 1600have tightly associated thereto third antibodies 1610 bound to thefluorophores 1540; the fluorophores 1540 can, if required, befluorophores bound in the aforesaid latex spheres.

[0390] In operation, the optical aperture 450 is interrogated usingevanescent radiation to obtain a first fluorescence measurement. Next, amucus and/or sputum sample is collected in the interior surface of thetube 200. The sample is then mechanically concentrated using the plunger230 as described in the foregoing and finally deposited onto the opticalaperture 450.

[0391] In STEP 2, the pathogens 1520 in the mechanically-concentratedsample have greater affinity for the first antibodies 1500 andcompetitively displace the analogues 1600 which become detached andmigrate with their associated fluorophores to regions remote from wherethe evanescent radiation propagates at the optical aperture 450. Theoptical aperture 450 is then interrogated for a second time withevanescent wave radiation of identical amplitude to that use to obtainthe first measurement; a second fluorescence measurement is therebyobtained. A difference between the first and second measurements isindicative of the number of displaced fluorophores and hence, byinference, the presence of the pathogen 1520 in the collected sample.

[0392] 4.6.3 Assay Detection Methods Not Involving Fluorescence orEvanescent Waves

[0393] The measurement system 10 can be adapted to utilize a detectionand labeling scheme that does not rely on evanescent wave excitation offluorescence. An example of such a scheme will be outlined: StandardMethod: STEP 1: Incubate sample with surface-bound IgG; any analytepresent is immobilised at surface by IgG STEP 2: Rinse STEP 3: Incubatewith labeled IgG. If any immobilised analyte is present, labeled IgG isimmobilised at surface STEP 4: Rinse to remove unbound IgG and labelSTEP 5: Add developing agent if required STEP 6: Measure result

[0394] With all such schemes, if monoclonal antibodies targeted at twodifferent epitopes are used, it is potentially possible to perform thetwo incubation steps simultaneously, thereby circumventing the need torinse between the at STEPS 2 and 4.

[0395] Possible labels include, but are not limited to, substanceslisted in Table 3. TABLE 3 Chemical labels Technique Label Developingagent Measurement Chromogenic chromogenic Enzyme substrate Bulkcolourimetry ELISA enzyme Fluorogenic ELISA fluorogenic enzyme Enzymesubstrate Bulk fluorescence Chemiluminescent chemiluminescentChemiluminescent Chemiluminescence assay molecule substrate at surfaceRadio immuno- Radio isotope None Radiation at surface assay (RIA)Colloidal gold Colloidal gold (optional) plating Colourimetry at surfaceassay solution to increase size of colloids

[0396] 5.0 Applications for Use of the Measurement System

[0397] The biological measurement system 10 can be used in applicationswhere the sample is not sputum and/or mucus. Possible other samples foranalysis by the system 10 include, for example, one or more of:

[0398] (a) blood;

[0399] (b) urine;

[0400] (c) pathogenic sera;

[0401] (d) semen;

[0402] (e) saliva;

[0403] (f) tears; and

[0404] (g) sweat.

[0405] Moreover, the measurement system 10 can also be adapted tointerrogate airborne particles such as airborne micro-organisms, spores,pollen, or airborne dust (for example from chemical processing plantswhere dangerous chemicals are used and/or manufactured).

[0406] The measurement system 10 described in the foregoing can beadapted for use in the detection of many other bacterial and viralinfections including, but not limited to:

[0407] (a) other forms of pneumonia such as influenzal pneumonia orviral pneumonia;

[0408] (b) tuberculosis;

[0409] (c) malaria;

[0410] (d) diptheria;

[0411] (e) lupuserethemytosis;

[0412] (f) pertussis;

[0413] (g) other zymotic diseases;

[0414] (h) streptococcus; and

[0415] (i) staphylococcus.

[0416] The measurement system 10 can also be applied to detect viralparticles, allergens or spores, pollen or other particles of abiological nature, or particles which are non-organic but can bedetected by antibodies, nucleic acids or other suitable recognitiongroups. Such non-organic particles can include airborne particles oftoxic compounds, controlled narcotics, explosives or any other particlesthat are present in air, water and other liquids.

[0417] Moreover, the measurement system 10 can be adapted to detectsympathetic particles such as indicators of certain forms of cancer.

[0418] Moreover, the measurement system 10 can be applied to detectparticles that do not cause disease, such as antibodies. Thus, themeasurement system 10 can be readily adapted for use in the earlydetection of HIV and AIDS, thereby being potentially valuable technologyfor countries such as South Africa which is having to cope with suchdiseases.

[0419] It will be appreciated that the aforementioned biologicalmeasurement system 10 can be modified. For example, although antibodiesare used to recognise and bind particles to be interrogated forpathogens, other recognition groups can be employed. For example, one ormore of the following substances can be used:

[0420] (a) proteins such as enzymes;

[0421] (b) aptamers of other sequences of nucleic acid or nucleic acidanalogues;

[0422] (c) analogues of proteins;

[0423] (d) artificial polypeptides; and

[0424] (e) entire organisms.

[0425] If the particles in the sample are themselves fluorescent, theycan be interrogated directly to generate the radiation; such particlescircumvent the need for treatment with fluorescently-labeled antibodiesas described in the foregoing.

1. A biological measurement system (10) for measuring the concentrationof components included in a sample, the system (10) characterised inthat it comprises: (a) collecting means (30) adapted for collecting thesample in aerosol form; (b) concentrating means (200, 230, 430) formechanically collecting the sample from the inside surface of thecollecting means; (c) marking means (1530, 1540) for optically labellingthe components present in the concentrated sample; and (d) interrogatingmeans for optically interrogating the labelled components and therebygenerating a measure of the concentration of components present in thesample.
 2. A system (10) according to claim 1, wherein the concentratingmeans further comprises a feature for scraping surfaces where the sampleis deposited to spatially concentrate the sample.
 3. A system. (10)according to claim 2, wherein the feature is elastically deformable forspreading the spatially concentrated sample over an opticalinterrogation region whereat the concentrated sample is subjected tooptical interrogation.
 4. A system (10) according to claim 1, whereinthe marking means comprises at least one of a selective binding assayand a competitive displacement assay for optically marking presence ofthe components by way of fluorescent markers; the fluorescent markersare bound to antibodies for use in at least one of the selective assayand the competitive assay; the fluorescent markers comprise fluorophoresbound to the antibodies by way of an intermediate carrier such that aplurality of fluorophores are associated with each antibody; and theintermediate carrier is implemented in the form of latex spheres.
 5. Asystem (10) according to claim 1, wherein the interrogating meanscomprises an optical evanescent detector (50, 800, 810) for detectingchanges in optical response induced by the presence of the components.6. A system (10) according to claim 5, wherein the evanescent detectorincludes: (a) one or more of a diode laser and a LED as a source ofinterrogating radiation for interrogating the concentrated sample; and(b) one or more of an avalanche photodiode, a photodiode array and aphotomultiplier tube as an optical detector for detecting fluorescentradiation emitted from the concentrated sample in response to opticalinterrogation of the sample, the optical detector for generating adetection signal indicative of changes in fluorescence from the sampleresulting from the presence of the components in the sample.
 7. A system(10) according to claim 1, wherein the collecting means (200, 230) isarranged to enclose the sample, whereby preventing personal contact withthe sample when the system is in use.
 8. A system (10) according toclaim 7, wherein the collection means (200, 230) can operate as a singleuse disposable part.
 9. A system (10) according to claim 8, wherein thecollecting means comprises features (1000) rendering it substantiallyunusable after sample collection therein.
 10. A system (10) according toclaim 1, wherein the collecting means (230) comprises vortex enhancingmeans (610, 650) for deposition of the sample within the collectingmeans.
 11. A system (10) according to claim 1, wherein the collectingmeans comprising a filtering means (105; 250) for at least partiallyinhibiting spread of the components of the sample from the collectingmeans.
 12. A system (10) according to claim 1, wherein the marking meansincludes lysing means for causing lysis of the components present in thesample, thereby enhancing measurement sensitivity of the system (10) byincreasing the number of available potential optical labelling sites.13. A method of detecting one or more pathogens in one or more samplesof the sputum/mucus from a subject using a system as claimed in any oneof the preceding claims, the method involving the steps of: (a)collecting said one or more samples in aerosol form in the collectingmeans; (b) spatially concentrating the one or more samples in theconcentrating means by mechanically collecting the sample from theinside surface of the collecting means; (c) optically labelling one ormore pathogens present in said-one or more samples; (d) opticallyinterrogating the pathogens to achieve an optical response; and (e)determining from the optical response of said one or more sampleswhether or not said one or more pathogens are present in said one ormore samples.
 14. A method according to claim 13, wherein the steps (b),(c) and (d), detection of the presence of a pathogen is performed usingevanescent-wave spectroscopy.
 15. A method according to either one ofclaims 13 and 14 adapted for the detection of bacteria associated withpulmonary and pulmonary-related infections.
 16. A method according toany one of claims 13 to 15, wherein the inhalation of one or more of:esters, eater vapour, saline vapour, expectorant and menthol is used toassist release of bacteria-containing mucus from the trachea or from theupper lung of a subject being tested.
 17. A method according to any oneof claims 13 to 15, wherein a partial negative pressure is employed toassist in obtaining said one or more samples in aerosol form.
 18. Amethod according to any one of claims 13 to 17, wherein said one or moresamples comprise an aerosol of blood or other bodily fluid or bodilyfluid in liquid form.
 19. A method according to any one of claims 13 to18, wherein analysis of said one or more samples is performed using onor both of: (a) an ELISA chromogenic reaction; and (b) a surfaceacoustic wave (SAW) biosensor to detect an antigen in said one or moresamples.